Nucleic acid and encoded zinc transporter protein entitled 108P5H8 useful in treatment and detection of cancer

ABSTRACT

A novel gene (designated 108P5H8) and its encoded protein, and variants thereof, are described wherein 108P5H8 exhibits tissue specific expression in normal adult tissue, and is aberrantly expressed in the cancers listed in Table I. Consequently, 108P5H8 provides a diagnostic, prognostic, prophylactic and/or therapeutic target for cancer. The 108P5H8 gene or fragment thereof, or its encoded protein, or variants thereof, or a fragment thereof, can be used to elicit a humoral or cellular immune response; antibodies or T cells reactive with 191P1E1B can be used in active or passive immunization.

RELATED APPLICATIONS

[0001] This application claims priority from provisional applicationU.S. Ser. No 60/256,210 filed Dec. 15, 2000, the contents of which areincorporated herein by reference.

FIELD OF THE INVENTION

[0002] The invention described herein relates to a gene and its encodedprotein, termed 108P5H8, expressed in certain cancers such as thoselisted in Table I, and to diagnostic, prognostic, prophylactic and/ortherapeutic methods and compositions useful in the management of cancersthat express 108P5H8.

BACKGROUND OF THE INVENTION

[0003] Cancer is the second leading cause of human death next tocoronary disease. Worldwide, millions of people die from cancer everyyear. In the United States alone, as reported by the American CancerSociety, cancer causes the death of well over a half-million peopleannually, with over 1.2 million new cases diagnosed per year. Whiledeaths from heart disease have been declining significantly, thoseresulting from cancer generally are on the rise. In the early part ofthe next century, cancer is predicted to become the leading cause ofdeath.

[0004] Worldwide, several cancers stand out as the leading killers. Inparticular, carcinomas of the lung, prostate, breast, colon, pancreas,and ovary represent the primary causes of cancer death. These andvirtually all other carcinomas share a common lethal feature. With veryfew exceptions, metastatic disease from a carcinoma is fatal. Moreover,even for those cancer patients who initially survive their primarycancers, common experience has shown that their lives are dramaticallyaltered. Many cancer patients experience strong anxieties driven by theawareness of the potential for recurrence or treatment failure. Manycancer patients experience physical debilitations following treatment.Furthermore, many cancer patients experience a recurrence.

[0005] Worldwide, prostate cancer is the fourth most prevalent cancer inmen. In North America and Northern Europe, it is by far the most commoncancer in males and is the second leading cause of cancer death in men.In the United States alone, well over 30,000 men die annually of thisdisease—second only to lung cancer. Despite the magnitude of thesefigures, there is still no effective treatment for metastatic prostatecancer. Surgical prostatectomy, radiation therapy, hormone ablationtherapy, surgical castration and chemotherapy continue to be the maintreatment modalities. Unfortunately, these treatments are ineffectivefor many and are often associated with undesirable consequences.

[0006] On the diagnostic front, the lack of a prostate tumor marker thatcan accurately detect early-stage, localized tumors remains asignificant limitation in the diagnosis and management of this disease.Although the serum prostate specific antigen (PSA) assay has been a veryuseful tool, however its specificity and general utility is widelyregarded as lacking in several important respects.

[0007] Progress in identifying additional specific markers for prostatecancer has been improved by the generation of prostate cancer xenograftsthat can recapitulate different stages of the disease in mice. The LAPC(Los Angeles Prostate Cancer) xenografts are prostate cancer xenograftsthat have survived passage in severe combined immune deficient (SCID)mice and have exhibited the capacity to mimic the transition fromandrogen dependence to androgen independence (Klein et al., 1997, Nat.Med. 3:402). More recently identified prostate cancer markers includePCTA-1 (Su et al., 1996, Proc. Natl. Acad. Sci. USA 93: 7252),prostate-specific membrane (PSM) antigen (Pinto et al., Clin Cancer ResSep. 2, 1996 (9): 1445-51), STEAP (Hubert, et al., Proc Natl Acad Sci US A. Dec. 7, 1999; 96(25): 14523-8) and prostate stem cell antigen(PSCA) (Reiter et al., 1998, Proc. Natl. Acad. Sci. USA 95: 1735).

[0008] While previously identified markers such as PSA, PSM, PCTA andPSCA have facilitated efforts to diagnose and treat prostate cancer,there is need for the identification of additional markers andtherapeutic targets for prostate and related cancers in order to furtherimprove diagnosis and therapy.

[0009] Renal cell carcinoma (RCC) accounts for approximately 3 percentof adult malignancies. Once adenomas reach a diameter of 2 to 3 cm,malignant potential exists. In the adult, the two principal malignantrenal tumors are renal cell adenocarcinoma and transitional cellcarcinoma of the renal pelvis or ureter. The incidence of renal celladenocarcinoma is estimated at more than 29,000 cases in the UnitedStates, and more than 11,600 patients died of this disease in 1998.Transitional cell carcinoma is less frequent, with an incidence ofapproximately 500 cases per year in the United States.

[0010] Surgery has been the primary therapy for renal celladenocarcinoma for many decades. Until recently, metastatic disease hasbeen refractory to any systemic therapy. With recent developments insystemic therapies, particularly immunotherapies, metastatic renal cellcarcinoma may be approached aggressively in appropriate patients with apossibility of durable responses. Nevertheless, there is a remainingneed for effective therapies for these patients.

[0011] Of all new cases of cancer in the United States, bladder cancerrepresents approximately 5 percent in men (fifth most common neoplasm)and 3 percent in women (eighth most common neoplasm). The incidence isincreasing slowly, concurrent with an increasing older population. In1998, there was an estimated 54,500 cases, including 39,500 in men and15,000 in women. The age-adjusted incidence in the United States is 32per 100,000 for men and 8 per 100,000 in women. The historic male/femaleratio of 3:1 may be decreasing related to smoking patterns in women.There were an estimated 11,000 deaths from bladder cancer in 1998 (7,800in men and 3,900 in women). Bladder cancer incidence and mortalitystrongly increase with age and will be an increasing problem as thepopulation becomes more elderly.

[0012] Most bladder cancers recur in the bladder. Bladder cancer ismanaged with a combination of transurethral resection of the bladder(TUR) and intravesical chemotherapy or immunotherapy. The multifocal andrecurrent nature of bladder cancer points out the limitations of TUR.Most muscle-invasive cancers are not cured by TUR alone. Radicalcystectomy and urinary diversion is the most effective means toeliminate the cancer but carry an undeniable impact on urinary andsexual function. There continues to be a significant need for treatmentmodalities that are beneficial for bladder cancer patients.

[0013] An estimated 130,200 cases of colorectal cancer occurred in 2000in the United States, including 93,800 cases of colon cancer and 36,400of rectal cancer. Colorectal cancers are the third most common cancersin men and women. Incidence rates declined significantly during1992-1996 (−2.1% per year).

[0014] Research suggests that these declines have been due to increasedscreening and polyp removal, preventing progression of polyps toinvasive cancers. There were an estimated 56,300 deaths (47,700 fromcolon cancer, 8,600 from rectal cancer) in 2000, accounting for about11% of all U.S. cancer deaths.

[0015] At present, surgery is the most common form of therapy forcolorectal cancer, and for cancers that have not spread, it isfrequently curative. Chemotherapy, or chemotherapy plus radiation, isgiven before or after surgery to most patients whose cancer has deeplyperforated the bowel wall or has spread to the lymph nodes. A permanentcolostomy (creation of an abdominal opening for elimination of bodywastes) is occasionally needed for colon cancer and is infrequentlyrequired for rectal cancer. There continues to be a need for effectivediagnostic and treatment modalities for colorectal cancer.

[0016] There were an estimated 164,100 new cases of lung and bronchialcancer in 2000, accounting for 14% of all U.S. cancer diagnoses. Theincidence rate of lung and bronchial cancer is declining significantlyin men, from a high of 86.5 per 100,000 in 1984 to 70.0 in 1996. In the1990s, the rate of increase among women began to slow. In 1996, theincidence rate in women was 42.3 per 100,000.

[0017] Lung and bronchial cancer caused an estimated 156,900 deaths in2000, accounting for 28% of all cancer deaths. During 1992-1996,mortality from lung cancer declined significantly among men (−1.7% peryear) while rates for women were still significantly increasing (0.9%per year). Since 1987, more women have died each year of lung cancerthan breast cancer, which, for over 40 years, was the major cause ofcancer death in women. Decreasing lung cancer incidence and mortalityrates most likely resulted from decreased smoking rates over theprevious 30 years; however, decreasing smoking patterns among women lagbehind those of men. Of concern, although the declines in adult tobaccouse have slowed, tobacco use in youth is increasing again.

[0018] Treatment options for lung and bronchial cancer are determined bythe type and stage of the cancer and include surgery, radiation therapy,and chemotherapy. For many localized cancers, surgery is usually thetreatment of choice. Because the disease has usually spread by the timeit is discovered, radiation therapy and chemotherapy are often needed incombination with surgery. Chemotherapy alone or combined with radiationis the treatment of choice for small cell lung cancer; on this regimen,a large percentage of patients experience remission, which in some casesis long lasting. There is however, an ongoing need for effectivetreatment and diagnostic approaches for lung and bronchial cancers.

[0019] An estimated 182,800 new invasive cases of breast cancer wereexpected to occur among women in the United States during 2000.Additionally, about 1,400 new cases of breast cancer were expected to bediagnosed in men in 2000. After increasing about 4% per year in the1980s, breast cancer incidence rates in women have leveled off in the1990s to about 110.6 cases per 100,000.

[0020] In the U.S. alone, there were an estimated 41,200 deaths (40,800women, 400 men) in 2000 due to breast cancer. Breast cancer ranks secondamong cancer deaths in women. According to the most recent data,mortality rates declined significantly during 1992-1996 with the largestdecreases in younger women, both white and black. These decreases wereprobably the result of earlier detection and improved treatment.

[0021] Taking into account the medical circumstances and the patient'spreferences, treatment of breast cancer may involve lumpectomy (localremoval of the tumor) and removal of the lymph nodes under the arm;mastectomy (surgical removal of the breast) and removal of the lymphnodes under the arm; radiation therapy; chemotherapy; or hormonetherapy. Often, two or more methods are used in combination. Numerousstudies have shown that, for early stage disease, long-term survivalrates after lumpectomy plus radiotherapy are similar to survival ratesafter modified radical mastectomy. Significant advances inreconstruction techniques provide several options for breastreconstruction after mastectomy. Recently, such reconstruction has beendone at the same time as the mastectomy.

[0022] Local excision of ductal carcinoma in situ (DCIS) with adequateamounts of surrounding normal breast tissue may prevent the localrecurrence of the DCIS. Radiation to the breast and/or tamoxifen mayreduce the chance of DCIS occurring in the remaining breast tissue. Thisis important because DCIS, if left untreated, may develop into invasivebreast cancer. Nevertheless, there are serious side effects or sequelaeto these treatments. There is, therefore, a need for efficacious breastcancer treatments.

[0023] There were an estimated 23,100 new cases of ovarian cancer in theUnited States in 2000. It accounts for 4% of all cancers among women andranks second among gynecologic cancers. During 1992-1996, ovarian cancerincidence rates were significantly declining. Consequent to ovariancancer, there were an estimated 14,000 deaths in 2000. Ovarian cancercauses more deaths than any other cancer of the female reproductivesystem.

[0024] Surgery, radiation therapy, and chemotherapy are treatmentoptions for ovarian cancer. Surgery usually includes the removal of oneor both ovaries, the fallopian tubes (salpingo-oophorectomy), and theuterus (hysterectomy). In some very early tumors, only the involvedovary will be removed, especially in young women who wish to havechildren. In advanced disease, an attempt is made to remove allintra-abdominal disease to enhance the effect of chemotherapy. Therecontinues to be an important need for effective treatment options forovarian cancer.

[0025] There were an estimated 28,300 new cases of pancreatic cancer inthe United States in 2000. Over the past 20 years, rates of pancreaticcancer have declined in men. Rates among women have remainedapproximately constant but may be beginning to decline. Pancreaticcancer caused an estimated 28,200 deaths in 2000 in the United States.Over the past 20 years, there has been a slight but significant decreasein mortality rates among men (about −0.9% per year) while rates haveincreased slightly among women.

[0026] Surgery, radiation therapy, and chemotherapy are treatmentoptions for pancreatic cancer. These treatment options can extendsurvival and/or relieve symptoms in many patients but are not likely toproduce a cure for most. There is a significant need for additionaltherapeutic and diagnostic options for pancreatic cancer.

SUMMARY OF THE INVENTION

[0027] The present invention relates to a gene, designated 108P5H8, thathas now been found to be over-expressed in the cancer(s) listed in TableI. Northern blot expression analysis of 108P5H8 gene expression innormal tissues shows a restricted expression pattern in adult tissues.The nucleotide (FIG. 2) and amino acid (FIG. 2, and FIG. 3) sequences of108P5H8 are provided. The tissue-related profile of 108P5H8 in normaladult tissues, combined with the over-expression observed in the tumorslisted in Table I, shows that 108P5H8 is aberrantly over-expressed in atleast some cancers, and thus serves as a useful diagnostic,prophylactic, prognostic, and/or therapeutic target for cancers of thetissue(s) such as those listed in Table I.

[0028] The invention provides polynucleotides corresponding orcomplementary to all or part of the 108P5H8 genes, mRNAs, and/or codingsequences, preferably in isolated form, including polynucleotidesencoding 108P5H8-related proteins and fragments of 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more than25 contiguous amino acids; at least 30, 35, 40, 45, 50, 55, 60, 65, 70,80, 85, 90, 95, 100 or more than 100 contiguous amino acids of a108P5H8-related protein, as well as the peptides/proteins themselves;DNA, RNA, DNA/RNA hybrids, and related molecules, polynucleotides oroligonucleotides complementary or having at least a 90% homology to the108P5H8 genes or mRNA sequences or parts thereof, and polynucleotides oroligonucleotides that hybridize to the 108P5H8 genes, mRNAs, or to108P5H8-encoding polynucleotides. Also provided are means for isolatingcDNAs and the genes encoding 108P5H8. Recombinant DNA moleculescontaining 108P5H8 polynucleotides, cells transformed or transduced withsuch molecules, and host-vector systems for the expression of 108P5H8gene products are also provided. The invention further providesantibodies that bind to 108P5H8 proteins and polypeptide fragmentsthereof, including polyclonal and monoclonal antibodies, murine andother mammalian antibodies, chimeric antibodies, humanized and fullyhuman antibodies, and antibodies labeled with a detectable marker ortherapeutic agent. In certain embodiments there is a proviso that theentire nucleic acid sequence of FIG. 2 is not encoded and/or the entireamino acid sequence of FIG. 2 is not prepared. In certain embodiments,the entire nucleic acid sequence of FIG. 2 is encoded and/or the entireamino acid sequence of FIG. 2 is prepared, either of which are inrespective human unit dose forms.

[0029] The invention further provides methods for detecting the presenceand status of 108P5H8 polynucleotides and proteins in various biologicalsamples, as well as methods for identifying cells that express 108P5H8.A typical embodiment of this invention provides methods for monitoring108P5H8 gene products in a tissue or hematology sample having orsuspected of having some form of growth dysregulation such as cancer.

[0030] The invention further provides various immunogenic or therapeuticcompositions and strategies for treating cancers that express 108P5H8such as cancers of tissues listed in Table I, including therapies aimedat inhibiting the transcription, translation, processing or function of108P5H8 as well as cancer vaccines. In one aspect, the inventionprovides compositions, and methods comprising them, for treating acancer that expresses 108P5H8 in a human subject wherein the compositioncomprises a carrier suitable for human use and a human unit dose of oneor more than one agent that inhibits the production or function of108P5H8. Preferably, the carrier is a uniquely human carrier. In anotheraspect of the invention, the agent is a moiety that is immunoreactivewith 108P5H8 protein. Non-limiting examples of such moieties include,but are not limited to, antibodies (such as single chain, monoclonal,polyclonal, humanized, chimeric, or human antibodies), functionalequivalents thereof (whether naturally occurring or synthetic), andcombinations thereof. The antibodies can be conjugated to a diagnosticor therapeutic moiety. In another aspect, the agent is a small moleculeas defined herein.

[0031] In another aspect, the agent comprises one or more than onepeptide which comprises a cytotoxic T lymphocyte (CTL) epitope thatbinds an HLA class I molecule in a human to elicit a CTL response to108P5H8 and/or one or more than one peptide which comprises a helper Tlymphocyte (HTL) epitope which binds an HLA class II molecule in a humanto elicit an HTL response. The peptides of the invention may be on thesame or on one or more separate polypeptide molecules. In a furtheraspect of the invention, the agent comprises one or more than onenucleic acid molecule that expresses one or more than one of the CTL orHTL response stimulating peptides as described above. In yet anotheraspect of the invention, the one or more than one nucleic acid moleculemay express a moiety that is immunologically reactive with 108P5H8 asdescribed above. The one or more than one nucleic acid molecule may alsobe, or encodes, a molecule that inhibits production of 108P5H8.Non-limiting examples of such molecules include, but are not limited to,those complementary to a nucleotide sequence essential for production of108P5H8 (e.g. antisense sequences or molecules that form a triple helixwith a nucleotide double helix essential for 108P5H8 production) or aribozyme effective to lyse 108P5H8 mRNA.

BRIEF DESCRIPTION OF THE FIGURES

[0032]FIG. 1. The 108P5H8 SSH sequence of 448 nucleotides.

[0033]FIG. 2. The cDNA and amino acid sequence of 108P5H8 v.1 is shownin FIG. 2A. The start methionine is underlined. The open reading frameextends from nucleic acid 253-1542 including the stop codon. The nucleicacid and amino acid sequence of 108P5H8 variant 2 is shown in FIG. 2B,the codon for the start methionine is underlined. The open reading framefor variant 2 extends from nucleic acid 1 to 1290 including the stopcodon. The nucleic acid and amino acid sequence of 108P5H8 variant 3 isshown in FIG. 2C, the codon for the start methionine is underlined. Theopen reading frame for variant 3 extends from nucleic acid 1-1290including the stop codon.

[0034]FIG. 3. Amino acid sequence of 108P5H8 variant 1 and of 108P5H8variant 2 is shown in FIG. 3A. The proteins encoded by the variant 1 andvariant 2 nucleic acid sequences are identical and each have 429 aminoacids. The amino acid sequence of 108P5H8 variant 3 is shown in FIG. 3B,the 108P5H8 v.3 protein has 429 amino acids.

[0035]FIG. 4. Amino acid alignments of 108P5H8 variants 1-3.

[0036]FIG. 5. Hydrophilicity amino acid profile of 108P5H8 determined bycomputer algorithm sequence analysis using the method of Hopp and Woods(Hopp T. P., Woods K. R., 1981. Proc. Natl. Acad. Sci. U.S.A.78:3824-3828) accessed on the Protscale website(www.expasy.ch/cgi-bin/protscale.p1) through the ExPasy molecularbiology server.

[0037]FIG. 6. Hydropathicity amino acid profile of 108P5H8 determined bycomputer algorithm sequence analysis using the method of Kyte andDoolittle (Kyte J., Doolittle R. F., 1982. J. Mol. Biol. 157:105-132)accessed on the ProtScale website (www.expasy.ch/cgi-bin/protscale.p1)through the ExPasy molecular biology server.

[0038]FIG. 7. Percent accessible residues amino acid profile of 108P5H8determined by computer algorithm sequence analysis using the method ofJanin (Janin J., 1979 Nature 277:491-492) accessed on the ProtScalewebsite (www.expasy.ch/cgi-bin/protscale.p1) through the ExPasymolecular biology server.

[0039]FIG. 8. Average flexibility amino acid profile of 108P5H8determined by computer algorithm sequence analysis using the method ofBhaskaran and Ponnuswamy (Bhaskaran R., and Ponnuswamy P. K., 1988. Int.J. Pept. Protein Res. 32:242-255) accessed on the ProtScale website(www.expasy.ch/cgi-bin/protscale.p1) through the ExPasy molecularbiology server.

[0040]FIG. 9. Beta-turn amino acid profile of 108P5H8 determined bycomputer algorithm sequence analysis using the method of Deleage andRoux (Deleage, G., Roux B. 1987 Protein Engineering 1:289-294) accessedon the ProtScale website (www.expasy.ch/cgi-bin/protscale.p1) throughthe ExPasy molecular biology server.

[0041]FIG. 10. Expression of 108P5H8 by RT-PCR. First strand cDNA wasprepared from vital pool 1 (VP1: liver, lung and kidney), vital pool 2(VP2, pancreas, colon and stomach), prostate xenograft pool (LAPC-4AD,LAPC-4AI, LAPC-9AD, LAPC-9AI), normal thymus, prostate cancer pool,bladder cancer pool, kidney cancer pool, colon cancer pool, lung cancerpool, ovary cancer pool, breast cancer pool, metastasis cancer pool,pancreas cancer pool, and from prostate cancer metastasis to lymph nodefrom 2 different patients. Normalization was performed by PCR usingprimers to actin and GAPDH. Semi-quantitative PCR using primers to108P5H8 was performed at 26 and 30 cycles of amplification. Strongexpression of 108P5H8 was observed in prostate cancer xenograft pool,prostate cancer pool and in the 2 different prostate cancer metastasissamples. Lower expression was detected in bladder cancer pool, kidneycancer pool, colon cancer pool, lung cancer pool, ovary cancer pool,breast cancer pool, metastasis pool, pancreas cancer pool, VP1 and VP2.

[0042]FIG. 11. Expression of 108P5H8 in normal human tissues and inprostate cancer xenografts. (A and B) Two multiple tissue Northernblots, with 2 mg of mRNA/lane, were probed with 108P5H8 sequence. Sizestandards in kilobases (kb) are indicated on the side. The results showstrong expression of an approximately 7 kb 108P5H8 transcript inprostate and lower expression in other tissues. (C) RNA was extractedfrom normal prostate, and from prostate cancer xenografts, LAPC-4AD,LAPC-4AI, LAPC-9AD, and LAPC-9AI. Northern blot with 10 mg of totalRNA/lane was probed with 108P5H8 sequence. Size standards in kilobases(kb) are indicated on the side. The results show expression of 108P5H8in all 4 xenografts and in normal prostate.

[0043]FIG. 12. Expression of 108P5H8 in prostate cancer xenografts. RNAwas extracted from prostate cancer xenografts, LAPC-4AD, and LAPC-9AD,injected either subcutaneously (sc) or intra-tibially (it) within themouse bone. LAPC-4 was also grown within a human bone implant in SCIDmice (LAPC-4 AD²). Northern blots with 10 μg of total RNA/lane wereprobed with the 108P5H8 SSH fragment. Size standards in kilobases (kb)are indicated on the side. Results show expression of 108P5H8 in allprostate cancer xenograft tissues tested.

[0044]FIG. 13. Expression of 108P5H8 in human cancer cell lines. RNA wasextracted from a panel of human cancer cell lines. Northern blots with10 mg of total RNA/lane were probed with the 108P5H8 SSH fragment. Sizestandards in kilobases (kb) are indicated on the side. Results show that108P5H8 is expressed in all cell lines tested such as prostate, bladder,brain, lung, kidney, breast, testis and ovary cancer cell lines.

[0045]FIG. 14. Expression of 108P5H8 in patient cancer specimens andcancer cell lines. Expression of 108P5H8 was assayed in a panel of humancancers (T) and their respective matched normal tissues (N) on RNA dotblots. 108P5H8 expression was seen in prostate, kidney, uterus andstomach cancers. The expression detected in some normal adjacent tissues(isolated from diseased tissues), but not in normal tissues (isolatedfrom healthy donors), may indicate that these tissues are not fullynormal and that 108P5H8 may be expressed in early-stage tumors. 108P5H8was also expressed in all 9 human cancer cell lines tested.

[0046]FIG. 15. Expression of 108P5H8 in prostate cancer patientspecimens. RNA was extracted from prostate tumors (T) and matched normaladjacent tissue (N_(AT)) isolated from prostate cancer patients.Northern blots with 10 mg of total RNA/lane were probed with 108P5H8sequence. Size standards in kilobases (kb) are indicated on the side.The results show expression of 108P5H8 in the two prostate tumors and inthe normal matched tissues.

[0047]FIG. 16. 108P5H8 is not Androgen-Regulated. LNCaP cells were grownin charcoal-stripped medium and stimulated with the synthetic androgenmibolerone, for either 14 or 24 hours. Northern blots with 10 mg oftotal RNA/lane were probed with either the 108P5H8 sequence (A), or withthe androgen-regulated gene PSA (B). A picture of the ethidium-bromidestaining of the RNA gel is also presented (C). Results show expressionof 108P5H8 is not regulated by androgen. The experimental samples wereconfirmed by testing for the expression of the androgen-regulatedprostate cancer gene PSA (B). This experiment shows that, as expected,PSA levels go down in presence of charcoal-stripped serum, andexpression is induced at 14 and 24 hours in presence of the syntheticandrogen.

[0048]FIG. 17. Expression of 108P5H8 in cancer metastasis patientspecimens. RNA was extracted from prostate cancer metastasis to lymphnode obtained from two different patient, as well as from normal bladder(NB), normal kidney (NK), normal lung (NL), normal breast (NBr), normalovary (NO), and normal pancreas (NPa). Northern blots with 10 mg oftotal RNA/lane were probed with 108P5H8 sequence. Size standards inkilobases (kb) are indicated on the side. The results show expression of108P5H8 in both cancer metastasis samples but not in normal tissues.

[0049]FIG. 18. Secondary structure and transmembrane prediction for108P5H8. The secondary structure of 108P5H8 protein was predicted usingthe HNN—Hierarchical Neural Network method (Guermeur, 1997,http://pbil.ibcp.fr/cgi-bin/npsa_automat.p1?page=npsa_nn.html), accessedfrom the ExPasy molecular biology server (http://www.expasy.ch/tools/).This method predicts the presence and location of alpha helices,extended strands, and random coils from the primary protein sequnce. Thepercent of the protein in a given secondary structure is also given.

[0050]FIG. 19. Transmembrane prediction for 108P5H8. A. Schematicrepresentation of the probability of existence of transmembrane regionsand orientation of 108P5H8 based on the TMpred algorithm of Hofmann andStoffel which utilizes TMBASE (K. Hofmann, W. Stoffel. TMBASE—A databaseof membrane spanning-protein segments Biol. Chem. Hoppe-Seyler 374:166,1993). B. Schematic representation of the probability of the existenceof transmembrane regions and the extracellular and intracellularorientation of 108P5H8 based on the TMHMM algorithm of Sonnhammer, vonHeijne, and Krogh (Erik L. L. Sonnhammer, Gunnar von Heijne, and AndersKrogh: A hidden Markov model for predicting transmembrane helices inprotein sequences. In Proc. of Sixth Int. Conf. on Intelligent Systemsfor Molecular Biology, p 175-182 Ed J. Glasgow, T. Littlejohn, F. Major,R. Lathrop, D. Sankoff, and C. Sensen Menlo Park, Calif.: AAAI Press,1998). The TMpred and TMHMM algorithms are accessed from the ExPasymolecular biology server (http://www.expasy.ch/tools/). The results ofthe transmembrane prediction programs presented in A and B depict108P5H8 as containing 6 transmembrane domains.

[0051]FIG. 20. Androgen-independent expression of 108P5H8 in prostatecancer cells. Western analysis of the indicated cell lysates werecarried out with a 1:2000 dilution of an anti-108P5H8 polyclonalantibody derived from immunization of a rabbit with a GST-fusion proteinencoding amino acids 1-112 of 108P5H8. 108P5H8 specific bands weredeveloped by incubation with an anti-rabbit HRP-conjugated secondaryantibody and visualized by enhanced chemiluminescence and exposure toautoradiography film. Indicated with an arrow is the full length 108P5H8protein. 293T cells overexpressing Myc His-tagged 108P5H8 serves as apositive control

[0052]FIG. 21. Surface expression of 108P5H8 in prostate cancer cells.LNCaP and LAPC4 cells were subjected to flow cytometric and fluorescencemicroscopic analysis of 108P5H8 expression using an anti-108P5H8polyclonal antibody or control rabbit IgG. Fluorescence was monitoredfollowing incubation with an FITC-conjugated anti-rabbit IgG secondaryantibody

[0053]FIG. 22. Surface expression of 108P5H8 in 293T cells. 293T cellswere transfected with either empty control vector or with pCDNA 3.1encoding the 108P5H8 cDNA and subjected to flow cytometry andfluorescence microscopy using an anti-108P5H8 polyclonal antibody (1:100dilution). Fluorescence was monitored following incubation with anFITC-conjugated anti-rabbit IgG secondary antibody. 293T-108P5H8 cellsexhibited strong surface fluorescence.

[0054]FIG. 23. Expression of 108P5H8 in prostate and ovarian cancerpatient specimens. Lysates from tumor (PCa) and normal adjacent tissue(NAT) from 2 prostate cancer patients and from a prostate cancermetastasis and tumor and normal adjacent tissue from an ovarian cancerpatient were subjected to Western analysis using anti-108P5H8 polyclonalantibody as described in FIG. 20. Indicated with an arrow is a 48 kDband representing full length 1-08P5H8. 108P5H8 protein was present inthe tumor tissue from the 2 prostate cancer patients and the metastasissample and in the normal adjacent tissue of 1 of the patients. 108P5H8was also expressed in the ovarian cancer sample but not in normal ovary.Low expression is seen in RNA positive 293T cells and strong expressionin the overexpressed 293T-108P5H8 cells.

[0055]FIG. 24. Expression of 108P5H8 in engineered cell lines. PC3 humanprostate cancer cells and NIH3T3 murine fibroblasts were engineered tostably express 108P5H8 through infection with retrovirus harboring the108P5H8 cDNA. Stable lines were generated by G418 selection for neomycinresistance. 108P5H8 expression was verified by Western blot analysiswith anti-108P5H8 polyclonal antibody as described in FIG. 20, using therespective cell lines expressing only the neomycin resistance gene asnegative controls.

[0056]FIG. 25. 108P5H8 protein variants show homology to human zinctransporter 4.

[0057]FIG. 26. Detection of 108P5H8 protein by immunocytochemistry inLNCaP cells. Immunocytochemical staining of LNCaP cells (an androgendependant prostate cancer cell line) showing expression of 108P5H8,which is not androgen regulated. LNCaP cell preparations were made fromeither cells grown in medium containing 10% fetal bovine serum (Panel A)or from cells grown for 72 hours in androgen free, serum depleted medium(by growing in charcoal dextran stripped medium) (Panel B) or frompreviously androgen starved cells which were subsequently stimulatedwith 10 mmol mibolerone, a synthetic androgen, for 48 hours (Panel C).LNCaP cells incubated with Rabbit IgG instead of rabbit antibody to108P5H8 was included to show no non-specific binding of rabbitimmunoglobulin to the cells (Panel D).

[0058]FIG. 27. Detection of 108P5H8 protein by immunohistochemistry inprostate cancer patient specimens. Immunohistochemical staining offrozen sections of a prostate carcinoma specimen (Gleason grade 6)showing expression of 108P5H8 in the neoplastic glands (Panel A) and nonon-specific binding of rabbit immunoglobulin in the Rabbit IgG control(Panel B).

[0059]FIG. 28. FIGS. 28A & 28B show a vertical alignment comparisonbetween nucleotide and amino acid sequences of the variants.

DETAILED DESCRIPTION OF THE INVENTION

[0060] Outline of Sections

[0061] I.) Definitions

[0062] II.) 108P5H8 Polynucleotides

[0063] II.A.) Uses of 108P5H8 Polynucleotides

[0064] II.A.1.) Monitoring of Genetic Abnormalities

[0065] II.A.2.) Antisense Embodiments

[0066] II.A.3.) Primers and Primer Pairs

[0067] II.A.4.) Isolation of 108P5H8-Encoding Nucleic Acid Molecules

[0068] II.A.5.) Recombinant Nucleic Acid Molecules and Host-VectorSystems

[0069] III.) 108P5H8-related Proteins

[0070] III.A.) Motif-bearing Protein Embodiments

[0071] III.B.) Expression of 108P5H8-related Proteins

[0072] III.C.) Modifications of 108P5H8-related Proteins

[0073] III.D.) Uses of 108P5H8-related Proteins

[0074] IV.) 108P5H8 Antibodies

[0075] V.) 108P5H8 Cellular Immune Responses

[0076] VI.) 108P5H8 Transgenic Animals

[0077] VII.) Methods for the Detection of 108P5H8

[0078] VIII.) Methods for Monitoring the Status of 108P5H8-related Genesand Their Products

[0079] IX.) Identification of Molecules That Interact With 108P5H8

[0080] X.) Therapeutic Methods and Compositions

[0081] X.A.) Anti-Cancer Vaccines

[0082] X.B.) 108P5H8 as a Target for Antibody-Based Therapy

[0083] X.C.) 108P5H8 as a Target for Cellular Immune Responses

[0084] X.C.1. Minigene Vaccines

[0085] X.C.2. Combinations of CTL Peptides with Helper Peptides

[0086] X.C.3. Combinations of CTL Peptides with T Cell Priming Agents

[0087] X.C.4. Vaccine Compositions Comprising DC Pulsed with CTL and/orHTL Peptides

[0088] X.D.) Adoptive Immunotherapy

[0089] X.E.) Administration of Vaccines for Therapeutic or ProphylacticPurposes

[0090] XI.) Diagnostic and Prognostic Embodiments of 108P5H8.

[0091] XII.) Inhibition of 108P5H8 Protein Function

[0092] XII.A.) Inhibition of 108P5H8 With Intracellular Antibodies

[0093] XII.B.) Inhibition of 108P5H8 with Recombinant Proteins

[0094] XII.C.) Inhibition of 108P5H8 Transcription or Translation

[0095] XII.D.) General Considerations for Therapeutic Strategies

[0096] XIII.) KITS

[0097] I.) Definitions:

[0098] Unless otherwise defined, all terms of art, notations and otherscientific terms or terminology used herein are intended to have themeanings commonly understood by those of skill in the art to which thisinvention pertains. In some cases, terms with commonly understoodmeanings are defined herein for clarity and/or for ready reference, andthe inclusion of such definitions herein should not necessarily beconstrued to represent a substantial difference over what is generallyunderstood in the art. Many of the techniques and procedures describedor referenced herein are well understood and commonly employed usingconventional methodology by those skilled in the art, such as, forexample, the widely utilized molecular cloning methodologies describedin Sambrook et al., Molecular Cloning: A Laboratory Manual 2nd. edition(1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. Asappropriate, procedures involving the use of commercially available kitsand reagents are generally carried out in accordance with manufacturerdefined protocols and/or parameters unless otherwise noted.

[0099] The terms “advanced prostate cancer”, “locally advanced prostatecancer”, “advanced disease” and “locally advanced disease” mean prostatecancers that have extended through the prostate capsule, and are meantto include stage C disease under the American Urological Association(AUA) system, stage C1-C2 disease under the Whitmore-Jewett system, andstage T3-T4 and N+ disease under the TNM (tumor, node, metastasis)system. In general, surgery is not recommended for patients with locallyadvanced disease, and these patients have substantially less favorableoutcomes compared to patients having clinically localized(organ-confined) prostate cancer. Locally advanced disease is clinicallyidentified by palpable evidence of induration beyond the lateral borderof the prostate, or asymmetry or induration above the prostate base.Locally advanced prostate cancer is presently diagnosed pathologicallyfollowing radical prostatectomy if the tumor invades or penetrates theprostatic capsule, extends into the surgical margin, or invades theseminal vesicles.

[0100] “Altering the native glycosylation pattern” is intended forpurposes herein to mean deleting one or more carbohydrate moieties foundin native sequence 108P5H8 (either by removing the underlyingglycosylation site or by deleting the glycosylation by chemical and/orenzymatic means), and/or adding one or more glycosylation sites that arenot present in the native sequence 108P5H8. In addition, the phraseincludes qualitative changes in the glycosylation of the nativeproteins, involving a change in the nature and proportions of thevarious carbohydrate moieties present.

[0101] The term “analog” refers to a molecule which is structurallysimilar or shares similar or corresponding attributes with anothermolecule (e.g. a 108P5H8-related protein). For example an analog of a108P5H8 protein can be specifically bound by an antibody or T cell thatspecifically binds to 108P5H8.

[0102] The term “antibody” is used in the broadest sense. Therefore an“antibody” can be naturally occurring or man-made such as monoclonalantibodies produced by conventional hybridoma technology. Anti-108P5H8antibodies comprise monoclonal and polyclonal antibodies as well asfragments containing the antigen-binding domain and/or one or morecomplementarity determining regions of these antibodies.

[0103] An “antibody fragment” is defined as at least a portion of thevariable region of the immunoglobulin molecule that binds to its target,i.e., the antigen-binding region. In one embodiment it specificallycovers single anti-108P5H8 antibodies and clones thereof (includingagonist, antagonist and neutralizing antibodies) and anti-108P5H8antibody compositions with polyepitopic specificity.

[0104] The term “codon optimized sequences” refers to nucleotidesequences that have been optimized for a particular host species byreplacing any codons having a usage frequency of less than about 20%.Nucleotide sequences that have been optimized for expression in a givenhost species by elimination of spurious polyadenylation sequences,elimination of exon/intron splicing signals, elimination oftransposon-like repeats and/or optimization of GC content in addition tocodon optimization are referred to herein as an “expression enhancedsequences.”

[0105] The term “cytotoxic agent” refers to a substance that inhibits orprevents the expression activity of cells, function of cells and/orcauses destruction of cells. The term is intended to include radioactiveisotopes chemotherapeutic agents, and toxins such as small moleculetoxins or enzymatically active toxins of bacterial, fungal, plant oranimal origin, including fragments and/or variants thereof. Examples ofcytotoxic agents include, but are not limited to maytansinoids, yttrium,bismuth, ricin, ricin A-chain, doxorubicin, daunorubicin, taxol,ethidium bromide, mitomycin, etoposide, tenoposide, vincristine,vinblastine, colchicine, dihydroxy anthracin dione, actinomycin,diphtheria toxin, Pseudomonas exotoxin (PE) A, PE40, abrin, abrin Achain, modeccin A chain, alpha-sarcin, gelonin, mitogellin,retstrictocin, phenomycin, enomycin, curicin, crotin, calicheamicin,sapaonaria officinalis inhibitor, and glucocorticoid and otherchemotherapeutic agents, as well as radioisotopes such as At²¹¹, I¹³¹,I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², P³² and radioactive isotopes ofLu. Antibodies may also be conjugated to an anti-cancer pro-drugactivating enzyme capable of converting the pro-drug to its active form.

[0106] The term “homolog” refers to a molecule which exhibits, homologyto another molecule, by for example, having sequences of chemicalresidues that are the same or similar at corresponding positions.

[0107] “Human Leukocyte Antigen” or “HLA” is a human class I or class IIMajor Histocompatibility Complex (MHC) protein (see, e.g., Stites, etal., IMMUNOLOGY, 8^(TH) ED., Lange Publishing, Los Altos, Calif. (1994).

[0108] The terms “hybridize”, “hybridizing”, “hybridizes” and the like,used in the context of polynucleotides, are meant to refer toconventional hybridization conditions, preferably such as hybridizationin 50% formamide/6×SSC/0.1% SDS/100 μg/ml ssDNA, in which temperaturesfor hybridization are above 37 degrees C. and temperatures for washingin 0.1×SSC/0.1% SDS are above 55 degrees C.

[0109] The phrases “isolated” or “biologically pure” refer to materialwhich is substantially or essentially free from components whichnormally accompany the material as it is found in its native state.Thus, isolated peptides in accordance with the invention preferably donot contain materials normally associated with the peptides in their insitu environment. For example, a polynucleotide is said to be “isolated”when it is substantially separated from contaminant polynucleotides thatcorrespond or are complementary to genes other than the 108P5H8 genes orthat encode polypeptides other than 108P5H8 gene product or fragmentsthereof. A skilled artisan can readily employ nucleic acid isolationprocedures to obtain an isolated 108P5H8 polynucleotide. A protein issaid to be “isolated,” for example, when physical, mechanical orchemical methods are employed to remove the 108P5H8 proteins fromcellular constituents that are normally associated with the protein. Askilled artisan can readily employ standard purification methods toobtain an isolated 108P5H8 protein. Alternatively, an isolated proteincan be prepared by chemical means.

[0110] The term “mammal” refers to any organism classified as a mammal,including mice, rats, rabbits, dogs, cats, cows, horses and humans. Inone embodiment of the invention, the mammal is a mouse. In anotherembodiment of the invention, the mammal is a human.

[0111] The terms “metastatic prostate cancer” and “metastatic disease”mean prostate cancers that have spread to regional lymph nodes or todistant sites, and are meant to include stage D disease under the AUAsystem and stage T×N×M+ under the TNM system. As is the case withlocally advanced prostate cancer, surgery is generally not indicated forpatients with metastatic disease, and hormonal (androgen ablation)therapy is a preferred treatment modality. Patients with metastaticprostate cancer eventually develop an androgen-refractory state within12 to 18 months of treatment initiation. Approximately half of theseandrogen-refractory patients die within 6 months after developing thatstatus. The most common site for prostate cancer metastasis is bone.Prostate cancer bone metastases are often osteoblastic rather thanosteolytic (i.e., resulting in net bone formation). Bone metastases arefound most frequently in the spine, followed by the femur, pelvis, ribcage, skull and humerus. Other common sites for metastasis include lymphnodes, lung, liver and brain. Metastatic prostate cancer is typicallydiagnosed by open or laparoscopic pelvic lymphadenectomy, whole bodyradionuclide scans, skeletal radiography, and/or bone lesion biopsy.

[0112] The term “monoclonal antibody” refers to an antibody obtainedfrom a population of substantially homogeneous antibodies, i.e., theantibodies comprising the population are identical except for possiblenaturally occurring mutations that are present in minor amounts.

[0113] A “motif”, as in biological motif of an 108P5H8-related protein,refers to any pattern of amino acids forming part of the primarysequence of a protein, that is associated with a particular function(e.g. protein-protein interaction, protein-DNA interaction, etc) ormodification (e.g. that is phosphorylated, glycosylated or amidated), orlocalization (e.g. secretory sequence, nuclear localization sequence,etc.) or a sequence that is correlated with being immunogenic, eitherhumorally or cellularly. A motif can be either contiguous or capable ofbeing aligned to certain positions that are generally correlated with acertain function or property. In the context of HLA motifs, “motif”refers to the pattern of residues in a peptide of defined length,usually a peptide of from about 8 to about 13 amino acids for a class IHLA motif and from about 6 to about 25 amino acids for a class II HLAmotif, which is recognized by a particular HLA molecule. Peptide motifsfor HLA binding are typically different for each protein encoded by eachhuman HLA allele and differ in the pattern of the primary and secondaryanchor residues.

[0114] A “pharmaceutical excipient” comprises a material such as anadjuvant, a carrier, pH-adjusting and buffering agents, tonicityadjusting agents, wetting agents, preservative, and the like.

[0115] “Pharmaceutically acceptable” refers to a non-toxic, inert,and/or composition that is physiologically compatible with humans orother mammals.

[0116] The term “polynucleotide” means a polymeric form of nucleotidesof at least 10 bases or base pairs in length, either ribonucleotides ordeoxynucleotides or a modified form of either type of nucleotide, and ismeant to include single and double stranded forms of DNA and/or RNA. Inthe art, this term if often used interchangeably with “oligonucleotide”.A polynucleotide can comprise a nucleotide sequence disclosed hereinwherein thymidine (T), as shown for example in FIG. 2, can also beuracil (U); this definition pertains to the differences between thechemical structures of DNA and RNA, in particular the observation thatone of the four major bases in RNA is uracil (U) instead of thymidine(T).

[0117] The term “polypeptide” means a polymer of at least about 4, 5, 6,7, or 8 amino acids. Throughout the specification, standard three letteror single letter designations for amino acids are used. In the art, thisterm is often used interchangeably with “peptide” or “protein”.

[0118] An HLA “primary anchor residue” is an amino acid at a specificposition along a peptide sequence which is understood to provide acontact point between the immunogenic peptide and the HLA molecule. Oneto three, usually two, primary anchor residues within a peptide ofdefined length generally defines a “motif” for an immunogenic peptide.These residues are understood to fit in close contact with peptidebinding groove of an HLA molecule, with their side chains buried inspecific pockets of the binding groove. In one embodiment, for example,the primary anchor residues for an HLA class I molecule are located atposition 2 (from the amino terminal position) and at the carboxylterminal position of a 8, 9, 10, 11, or 12 residue peptide epitope inaccordance with the invention. In another embodiment, for example, theprimary anchor residues of a peptide that will bind an HLA class IImolecule are spaced relative to each other, rather than to the terminiof a peptide, where the peptide is generally of at least 9 amino acidsin length. The primary anchor positions for each motif and supermotifare set forth in Table IV. For example, analog peptides can be createdby altering the presence or absence of particular residues in theprimary and/or secondary anchor positions shown in Table IV. Suchanalogs are used to modulate the binding affinity and/or populationcoverage of a peptide comprising a particular HLA motif or supermotif.

[0119] A “recombinant” DNA or RNA molecule is a DNA or RNA molecule thathas been subjected to molecular manipulation in vitro.

[0120] Non-limiting examples of small molecules include compounds thatbind or interact with 108P5H8, ligands including hormones,neuropeptides, chemokines, odorants, phospholipids, and functionalequivalents thereof that bind and preferably inhibit 108P5H8 proteinfunction. Such non-limiting small molecules preferably have a molecularweight of less than about 10 kDa, more preferably below about 9, about8, about 7, about 6, about 5 or about 4 kDa. In certain embodiments,small molecules physically associate with, or bind, 108P5H8 protein; arenot found in naturally occurring metabolic pathways; and/or are moresoluble in aqueous than non-aqueous solutions

[0121] “Stringency” of hybridization reactions is readily determinableby one of ordinary skill in the art, and generally is an empiricalcalculation dependent upon probe length, washing temperature, and saltconcentration. In general, longer probes require higher temperatures forproper annealing, while shorter probes need lower temperatures.Hybridization generally depends on the ability of denatured nucleic acidsequences to reanneal when complementary strands are present in anenvironment below their melting temperature. The higher the degree ofdesired homology between the probe and hybridizable sequence, the higherthe relative temperature that can be used. As a result, it follows thathigher relative temperatures would tend to make the reaction conditionsmore stringent, while lower temperatures less so. For additional detailsand explanation of stringency of hybridization reactions, see Ausubel etal., Current Protocols in Molecular Biology, Wiley IntersciencePublishers, (1995).

[0122] “Stringent conditions” or “high stringency conditions”, asdefined herein, are identified by, but not limited to, those that: (1)employ low ionic strength and high temperature for washing, for example0.015 M sodium chloride/0.0015 M sodium citrate/0.1% sodium dodecylsulfate at 50° C.; (2) employ during hybridization a denaturing agent,such as formamide, for example, 50% (v/v) formamide with 0.1% bovineserum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM sodiumphosphate buffer at pH 6.5 with 750 mM sodium chloride, 75 mM sodiumcitrate at 42° C.; or (3) employ 50% formamide, 5×SSC (0.75 M NaCl,0.075 M sodium citrate),50 mM sodium phosphate (pH 6.8), 0.1%sodiumtpyrophosphate, 5× Denhardt's solution, sonicated salmon sperm DNA(50 μg/ml), 0.1% SDS, and 10% dextran sulfate at 42° C., with washes at42° C. in 0.2×SSC (sodium chloride/sodium. citrate) and 50% formamide at55° C., followed by a high-stringency wash consisting of 0.1×SSCcontaining EDTA at 55° C. “Moderately stringent conditions” aredescribed by, but not limited to, those in Sambrook et al., MolecularCloning: A Laboratory Manual, New York: Cold Spring Harbor Press, 1989,and include the use of washing solution and hybridization conditions(e.g., temperature, ionic strength and % SDS) less stringent than thosedescribed above. An example of moderately stringent conditions isovernight incubation at 37° C. in a solution comprising: 20% formamide,5×SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH7.6), 5× Denhardt's solution, 10% dextran sulfate, and 20 mg/mLdenatured sheared salmon sperm DNA, followed by washing the filters in1×SSC at about 37-50° C. The skilled artisan will recognize how toadjust the temperature, ionic strength, etc. as necessary to accommodatefactors such as probe length and the like.

[0123] An HLA “supermotif” is a peptide binding specificity shared byHLA molecules encoded by two or more HLA alleles.

[0124] As used herein “to treat” or “therapeutic” and grammaticallyrelated terms, refer to any improvement of any consequence of disease,such as prolonged survival, less morbidity, and/or a lessening of sideeffects which are the byproducts of an alternative therapeutic modality;full eradication of disease is not required.

[0125] A “transgenic animal” (e.g., a mouse or rat) is an animal havingcells that contain a transgene, which transgene was introduced into theanimal or an ancestor of the animal at a prenatal, e.g., an embryonicstage. A “transgene” is a DNA that is integrated into the genome of acell from which a transgenic animal develops.

[0126] As used herein, an HLA or cellular immune response “vaccine” is acomposition that contains or encodes one or more peptides of theinvention. There are numerous embodiments of such vaccines, such as acocktail of one or more individual peptides; one or more peptides of theinvention comprised by a polyepitopic peptide; or nucleic acids thatencode such individual peptides or polypeptides, e.g., a minigene. thatencodes a polyepitopic peptide. The “one or more peptides” can includeany whole unit integer from 1-150 or more, e.g.; at least 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100,105, 110, 115, 120, 125, 130, 135, 140, 145, or 150 or more peptides ofthe invention. The peptides or polypeptides can optionally be modified,such as by lipidation, addition of targeting or other sequences. HLAclass I peptides of the invention can be admixed with, or linked to, HLAclass II peptides, to facilitate activation of both cytotoxic Tlymphocytes and helper T lymphocytes. HLA vaccines can also comprisepeptide-pulsed antigen presenting cells, e.g., dendritic cells.

[0127] The term “variant” refers to a molecule that exhibits a variationfrom a described type or norm, such as a protein that has one or moredifferent amino acid residues in the corresponding position(s) of aspecifically described protein (e.g. the 108P5H8 protein shown in FIG. 2or FIG. 3. An analog is an example of a variant protein. Splice isoformsand single nucleotides polymorphisms (SNPs) are further examples ofvariants.

[0128] The “108P5H8-related proteins” of the invention include thosespecifically identified herein, as well as allelic variants,conservative substitution variants, analogs and homologs that can beisolated/generated and characterized without undue experimentationfollowing the methods outlined herein or readily available in the art.Fusion proteins that combine parts of different 108P5H8 proteins orfragments thereof, as well as fusion proteins of a 108P5H8 protein and aheterologous polypeptide are also included. Such 108P5H8 proteins arecollectively referred to as the 108P5H8-related proteins, the proteinsof the invention, or 108P5H8. The term “108P5H8-related protein” refersto a polypeptide fragment or an 108P5H8 protein sequence of 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, ormore than 25 amino acids; or, at least 30, 35, 40, 45, 50, 55, 60, 65,70, 80, 85, 90, 95, 100 or more than 100 amino acids.

[0129] II.) 108P5H8 Polynucleotides

[0130] One aspect of the invention provides polynucleotidescorresponding or complementary to all or part of an 108P5H8 gene, mRNA,and/or coding sequence, preferably in isolated form, includingpolynucleotides encoding an 108P5H8-related protein and fragmentsthereof, DNA, RNA, DNA/RNA hybrid, and related molecules,polynucleotides or oligonucleotides complementary to an 108P5H8 gene ormRNA sequence or a part thereof, and polynucleotides or oligonucleotidesthat hybridize to an 108P5H8 gene, mRNA, or to an 108P5H8 encodingpolynucleotide, (collectively, “108P5H8 polynucleotides”). In allinstances when referred to in this section, T can also be U in FIG. 2.

[0131] Embodiments of a 108P5H8 polynucleotide include: a 108P5H8polynucleotide having the sequence shown in FIG. 2, the nucleotidesequence of 108P5H8 as shown in FIG. 2 wherein T is U; at least 10contiguous nucleotides of a polynucleotide having the sequence as shownin FIG. 2; or, at least 10 contiguous nucleotides of a polynucleotidehaving the sequence as shown in FIG. 2 where T is U. For example,embodiments of 108P5H8 nucleotides comprise, without limitation:

[0132] (I) a polynucleotide comprising, consisting essentially of, orconsisting of a sequence as shown in FIG. 2 (SEQ ID Nos.: 2569, 2571, &2573), wherein T can also be U;

[0133] (II) a polynucleotide comprising, consisting essentially of, orconsisting of the sequence as shown in FIG. 2A (SEQ ID NO.: 2569), fromnucleotide residue number 253 through nucleotide residue number 1542,wherein T can also be U;

[0134] (III) a polynucleotide comprising, consisting essentially of, orconsisting of the sequence as shown in FIG. 2B (SEQ ID NO.: 2571), fromnucleotide residue number 1 through nucleotide residue number 1290,wherein T can also be U;

[0135] (IV) a polynucleotide comprising, consisting essentially of, orconsisting of the sequence as shown in FIG. 2C (SEQ ID NO.: 2573), fromnucleotide residue number 1 through nucleotide residue number 1290,wherein T can also be U;

[0136] (V) a polynucleotide that encodes an 108P5H8-related protein thatis at least 90% homologous to an entire amino acid sequence shown inFIGS. 2A-C (SEQ ID Nos.: 2570, 2572, & 2574);

[0137] (VI) a polynucleotide that encodes an 108P5H8-related proteinthat is at least 90% identical to an entire amino acid sequence shown inFIGS. 2A-C (SEQ ID Nos.: 2570, 2572, & 2574);

[0138] (VII) a polynucleotide that encodes at least one peptide setforth in Tables V-XVIII, XXII, and XXIII;

[0139] (VIII) a polynucleotide that encodes a peptide region of at least5 amino acids of a peptide of FIGS. 3A or 3B in any whole numberincrement up to 429 that includes an amino acid position having a valuegreater than 0.5 in the Hydrophilicity profile of FIG. 5;

[0140] (IX) a polynucleotide that encodes a peptide region of at least 5amino acids of a peptide of FIGS. 3A or 3B in any whole number incrementup to 429 that includes an amino acid position having a value less than0.5 in the Hydropathicity profile of FIG. 6;

[0141] (X) a polynucleotide that encodes a peptide region of at least 5amino acids of a peptide of FIGS. 3A or 3B in any whole number incrementup to 429 that includes an amino acid position having a value greaterthan 0.5 in the Percent Accessible Residues profile of FIG. 7;

[0142] (XI) a polynucleotide that encodes a peptide region of at least 5amino acids of a peptide of FIGS. 3A or 3B in any whole number incrementup to 429 that includes an amino acid position having a value greaterthan 0.5 in the Average Flexibility profile on FIG. 8;

[0143] (XII) a polynucleotide that encodes a peptide region of at least5 amino acids of a peptide of FIGS. 3A or 3B in any whole numberincrement up to 429 that includes an amino acid position having a valuegreater than 0.5 in the Beta-turn profile of FIG. 9;

[0144] (XIII) a polynucleotide that encodes a 108P5H8-related proteinwhose sequence is encoded by the cDNAs contained in the plasmiddesignated p108P5H8-C deposited with American Type Culture Collection asAccession No. PTA-2198;

[0145] (XIV) a polynucleotide that is fully complementary to apolynucleotide of any one of (I)-(XIII);

[0146] (XV) a polynucleotide that selectively hybridizes under stringentconditions to a polynucleotide of (I)-(XIV);

[0147] (XVI) a peptide that is encoded by any of (I)-(XIII); and,

[0148] (XLII) a polynucleotide of any of (I)-(XV) or peptide of (XVI)together with a pharmaceutical excipient and/or in a human unit doseform.

[0149] As used herein, a range is understood to specifically discloseall whole unit positions thereof.

[0150] Typical embodiments of the invention disclosed herein include108P5H8 polynucleotides that encode specific portions of 108P5H8 mRNAsequences (and those which are complementary to such sequences) such asthose that encode the proteins and/or fragments thereof, for example:

[0151] 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95,100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165,170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 250, 275,300, 325, 350, 375, 400, 425, or 429 contiguous amino acids of variants1, 2 or 3.

[0152] For example, representative embodiments of the inventiondisclosed herein include: polynucleotides and their encoded-peptidesthemselves encoding about amino acid 1 to about amino acid 10 of the108P5H8 protein shown in FIG. 2 or FIG. 3, polynucleotides encodingabout amino acid 10 to about amino acid 20 of the 108P5H8 protein shownin FIG. 2, or FIG. 3, polynucleotides encoding about amino acid 20 toabout amino acid 30 of the 108P5H8 protein shown in FIG. 2 or FIG. 3,polynucleotides encoding about amino acid 30 to about amino acid 40 ofthe 108P5H8 protein shown in FIG. 2 or FIG. 3, polynucleotides encodingabout amino acid 40 to about amino acid 50 of the 108P5H8 protein shownin FIG. 2 or FIG. 3, polynucleotides encoding about amino acid 50 toabout amino acid 60 of the 108P5H8 protein shown in FIG. 2 or FIG. 3,polynucleotides encoding about amino acid 60 to about amino acid 70 ofthe 108P5H8 protein shown in FIG. 2 or FIG. 3, polynucleotides encodingabout amino acid 70 to about amino acid 80 of the 108P5H8 protein shownin FIG. 2 or FIG. 3, polynucleotides encoding about amino acid 80 toabout amino acid 90 of the 108P5H8 protein shown in FIG. 2 or FIG. 3,polynucleotides encoding about amino acid 90 to about amino acid 100 ofthe 108P5H8 protein shown in FIG. 2 or FIG. 3, in increments of about 10amino acids, ending at the carboxyl terminal amino acid set forth inFIG. 2 or FIG. 3. Accordingly polynucleotides encoding portions of theamino acid sequence (of about 10 amino acids), of amino acids 100through the carboxyl terminal amino acid of the 108P5H8 protein areembodiments of the invention. Wherein it is understood that eachparticular amino acid position discloses that position plus or minusfive amino acid residues.

[0153] Polynucleotides encoding relatively long portions of a 108P5H8protein are also within the scope of the invention. For example,polynucleotides encoding from about amino acid 1 (or 20 or 30 or 40etc.) to about amino acid 20, (or 30, or 40 or 50 etc.) of the 108P5H8protein shown in FIG. 2 or FIG. 3 can be generated by a variety oftechniques well known in the art. These polynucleotide fragments caninclude any portion of the 108P5H8 sequence as shown in FIG. 2.

[0154] Additional illustrative embodiments of the invention disclosedherein include 108P5H8 polynucleotide fragments encoding one or more ofthe biological motifs contained within a 108P5H8 protein sequence,including one or more of the motif-bearing subsequences of a 108P5H8protein set forth in Tables V-XVIII, XXII, and XXIII. In anotherembodiment, typical polynucleotide fragments of the invention encode oneor more of the regions of 108P5H8 that exhibit homology to a knownmolecule. In another embodiment of the invention, typical polynucleotidefragments can encode one or more of the 108P5H8 N-glycosylation sites,cAMP and cGMP-dependent protein kinase phosphorylation sites, caseinkinase II phosphorylation sites or N-myristoylation site and amidationsites.

[0155] II.A.) Uses of 108P5H8 Polynucleotides

[0156] II.A.1.) Monitoring of Genetic Abnormalities

[0157] The polynucleotides of the preceding paragraphs have a number ofdifferent specific uses. The human 108P5H8 gene maps to the chromosomallocation set forth in Example 3. For example, because the 108P5H8 genemaps to this chromosome, polynucleotides that encode different regionsof the 108P5H8 proteins are used to characterize cytogeneticabnormalities of this chromosomal locale, such as abnormalities that areidentified as being associated with various cancers. In certain genes, avariety of chromosomal abnormalities including rearrangements have beenidentified as frequent cytogenetic abnormalities in a number ofdifferent cancers (see e.g. Krajinovic et al., Mutat. Res. 382(3-4):81-83 (1998); Johansson et al., Blood 86(10): 3905-3914 (1995) andFinger et al., P.N.A.S. 85(23): 9158-9162 (1988)). Thus, polynucleotidesencoding specific regions of the 108P5H8 proteins provide new tools thatcan be used to delineate, with greater precision than previouslypossible, cytogenetic abnormalities in the chromosomal region thatencodes 108P5H8 that may contribute to the malignant phenotype. In thiscontext, these polynucleotides satisfy a need in the art for expandingthe sensitivity of chromosomal screening in order to identify moresubtle and less common chromosomal abnormalities (see e.g. Evans et al.,Am. J. Obstet. Gynecol 171(4): 1055-1057 (1994)).

[0158] Furthermore, as 108P5H8 was shown to be highly expressed inbladder and other cancers, 108P5H8 polynucleotides are used in methodsassessing the status of 108P5H8 gene products in normal versus canceroustissues. Typically, polynucleotides that encode specific regions of the108P5H8 proteins are used to assess the presence of perturbations (suchas deletions, insertions, point mutations, or alterations resulting in aloss of an antigen etc.) in specific regions of the 108P5H8 gene, suchas regions containing one or more motifs. Exemplary assays include bothRT-PCR assays as well as single-strand conformation polymorphism (SSCP)analysis (see, e.g., Marrogi et al., J. Cutan. Pathol. 26(8): 369-378(1999), both of which utilize polynucleotides encoding specific regionsof a protein to examine these regions within the protein.

[0159] II.A.2.) Antisense Embodiments

[0160] Other specifically contemplated nucleic acid related embodimentsof the invention disclosed herein are genomic DNA, cDNAs, ribozymes, andantisense molecules, as well as nucleic acid molecules based on analternative backbone, or including alternative bases, whether derivedfrom natural sources or synthesized, and include molecules capable ofinhibiting the RNA or protein expression of 108P5H8. For example,antisense molecules can be RNAs or other molecules, including peptidenucleic acids (PNAs) or non-nucleic acid molecules such asphosphorothioate derivatives, that specifically bind DNA or RNA in abase pair-dependent manner. A skilled artisan can readily obtain theseclasses of nucleic acid molecules using the 108P5H8 polynucleotides andpolynucleotide sequences disclosed herein.

[0161] Antisense technology entails the administration of exogenousoligonucleotides that bind to a target polynucleotide located within thecells. The term “antisense” refers to the fact that sucholigonucleotides are complementary to their intracellular targets, e.g.,108P5H8. See for example, Jack Cohen, Oligodeoxynucleotides, AntisenseInhibitors of Gene Expression, CRC Press, 1989; and Synthesis 1:1-5(1988). The 108P5H8 antisense oligonucleotides of the present inventioninclude derivatives such as S-oligonucleotides (phosphorothioatederivatives or S-oligos, see, Jack Cohen, supra), which exhibit enhancedcancer cell growth inhibitory action. S-oligos (nucleosidephosphorothioates) are isoelectronic analogs of an oligonucleotide(O-oligo) in which a nonbridging oxygen atom of the phosphate group isreplaced by a sulfur atom. The S-oligos of the present invention can beprepared by treatment of the corresponding O-oligos with3H-1,2-benzodithiol-3-one-1,1-dioxide, which is a sulfur transferreagent. See, e.g., Iyer, R. P. et al., J. Org. Chem. 55:4693-4698(1990); and Iyer, R. P. et al., J. Am. Chem. Soc. 112:1253-1254 (1990).Additional 108P5H8 antisense oligonucleotides of the present inventioninclude morpholino antisense oligonucleotides known in the art (see,e.g., Partridge et al., 1996, Antisense & Nucleic Acid Drug Development6: 169-175).

[0162] The 108P5H8 antisense oligonucleotides of the present inventiontypically can be RNA or DNA that is complementary to and stablyhybridizes with the first 100 5′ codons or last 100 3′ codons of a108P5H8 genomic sequence or the corresponding mRNA. Absolutecomplementarity is not required, although high degrees ofcomplementarity are preferred. Use of an oligonucleotide complementaryto this region allows for the selective hybridization to 108P5H8 mRNAand not to mRNA specifying other regulatory subunits of protein kinase.In one embodiment, 108P5H8 antisense oligonucleotides of the presentinvention are 15 to 30 -mer fragments of the antisense DNA molecule thathave a sequence that hybridizes to 108P5H8 mRNA. Optionally, 108P5H8antisense oligonucleotide is a 30 -mer oligonucleotide that iscomplementary to a region in the first 10 5′ codons or last 10 3′ codonsof 108P5H8. Alternatively, the antisense molecules are modified toemploy ribozymes in the inhibition of 108P5H8 expression, see, e.g., L.A. Couture & D. T. Stinchcomb; Trends Genet 12: 510-515 (1996).

[0163] II.A.3.) Primers and Primer Pairs

[0164] Further specific embodiments of this nucleotides of the inventioninclude primers and primer pairs, which allow the specific amplificationof polynucleotides of the invention or of any specific parts thereof,and probes that selectively or specifically hybridize to nucleic acidmolecules of the invention or to any part thereof. Probes can be labeledwith a detectable marker, such as, for example, a radioisotope,fluorescent compound, bioluminescent compound, a chemiluminescentcompound, metal chelator or enzyme. Such probes and primers are used todetect the presence of a 108P5H8 polynucleotide in a sample and as ameans for detecting a cell expressing a 108P5H8 protein.

[0165] Examples of such probes include polypeptides comprising all orpart of the human 108P5H8 cDNA sequence shown in FIG. 2. Examples ofprimer pairs capable of specifically amplifying 108P5H8 mRNAs are alsodescribed in the Examples. As will be understood by the skilled artisan,a great many different primers and probes can be prepared based on thesequences provided herein and used effectively to amplify and/or detecta 108P5H8 mRNA.

[0166] The 108P5H8 polynucleotides of the invention are useful for avariety of purposes, including but not limited to their use as probesand primers for the amplification and/or detection of the 108P5H8gene(s), mRNA(s), or fragments thereof; as reagents for the diagnosisand/or prognosis of prostate cancer and other cancers; as codingsequences capable of directing the expression of 108P5H8 polypeptides;as tools for modulating or inhibiting the expression of the 108P5H8gene(s) and/or translation of the 108P5H8 transcript(s); and astherapeutic agents.

[0167] The present invention includes the use of any probe as describedherein to identify and isolate a 108P5H8 or 108P5H8 related nucleic acidsequence from a naturally occurring source, such as humans or othermammals, as well as the isolated nucleic acid sequence per se, whichwould comprise all or most of the sequences found in the probe used.

[0168] II.A.4.) Isolation of 108P5H8-Encoding Nucleic Acid Molecules

[0169] The 108P5H8 cDNA sequences described herein enable the isolationof other polynucleotides encoding 108P5H8 gene product(s), as well asthe isolation of polynucleotides encoding 108P5H8 gene product homologs,alternatively spliced isoforms, allelic variants, and mutant forms of a108P5H8 gene product as well as polynucleotides that encode analogs of108P5H8-related proteins. Various molecular cloning methods that can beemployed to isolate full length cDNAs encoding an 108P5H8 gene are wellknown (see, for example, Sambrook, J. et al, Molecular Cloning: ALaboratory Manual, 2d edition, Cold Spring Harbor Press, New York, 1989;Current Protocols in Molecular Biology. Ausubel et al., Eds., Wiley andSons, 1995). For example, lambda phage cloning methodologies can beconveniently employed, using commercially available cloning systems(e.g., Lambda ZAP Express, Stratagene). Phage clones containing 108P5H8gene cDNAs can be identified by probing with a labeled 108P5H8 cDNA or afragment thereof. For example, in one embodiment, a 108P5H8 cDNA (e.g.,FIG. 2) or a portion thereof can be synthesized and used as a probe toretrieve overlapping and full-length cDNAs corresponding to a 108P5H8gene. A 108P5H8 gene itself can be isolated by screening genomic DNAlibraries, bacterial artificial chromosome libraries (BACs), yeastartificial chromosome libraries (YACs), and the like, with 108P5H8 DNAprobes or primers.

[0170] II.A.5.) Recombinant Nucleic Acid Molecules and Host-VectorSystems

[0171] The invention also provides recombinant DNA or RNA moleculescontaining an 108P5H8 polynucleotide, a fragment, analog or homologuethereof, including but not limited to phages, plasmids, phagemids,cosmids, YACs, BACs, as well as various viral and non-viral vectors wellknown in the art, and cells transformed or transfected with suchrecombinant DNA or RNA molecules. Methods for generating such moleculesare well known (see, for example, Sambrook et al., 1989, supra).

[0172] The invention further provides a host-vector system comprising arecombinant DNA molecule containing a 108P5H8 polynucleotide, fragment,analog or homologue thereof within a suitable prokaryotic or eukaryotichost cell. Examples of suitable eukaryotic host cells include a yeastcell, a plant cell, or an animal cell, such as a mammalian cell or aninsect cell (e.g., a baculovirus-infectible cell such as an Sf9 orHighFive cell). Examples of suitable mammalian cells include variousprostate cancer cell lines such as DU145 and TsuPr1, other transfectableor transducible prostate cancer cell lines, primary cells (PrEC), aswell as a number of mammalian cells routinely used for the expression ofrecombinant proteins (e.g., COS, CHO, 293, 293T cells). Moreparticularly, a polynucleotide comprising the coding sequence of 108P5H8or a fragment, analog or homolog thereof can be used to generate 108P5H8proteins or fragments thereof using any number of host-vector systemsroutinely used and widely known in the art.

[0173] A wide range of host-vector systems suitable for the expressionof 108P5H8 proteins or fragments thereof are available, see for example,Sambrook et al., 1989, supra; Current Protocols in Molecular Biology,1995, supra). Preferred vectors for mammalian expression include but arenot limited to pcDNA 3.1 myc-His-tag (Invitrogen) and the retroviralvector pSRαtkneo (Muller et al., 1991, MCB 11:1785). Using theseexpression vectors, 108P5H8 can be expressed in several prostate cancerand non-prostate cell lines, including for example 293, 293T, rat-1, NIH3T3 and TsuPr1. The host-vector systems of the invention are useful forthe production of a 108P5H8 protein or fragment thereof. Suchhost-vector systems can be employed to study the functional propertiesof 108P5H8 and 108P5H8 mutations or analogs.

[0174] Recombinant human 108P5H8 protein or an analog or homolog orfragment thereof can be produced by mammalian cells transfected with aconstruct encoding a 108P5H8-related nucleotide. For example, 293T cellscan be transfected with an expression plasmid encoding 108P5H8 orfragment, analog or homolog thereof, a 108P5H8-related protein isexpressed in the 293T cells, and the recombinant 108P5H8 protein isisolated using standard purification methods (e.g., affinitypurification using anti-108P5H8 antibodies). In another embodiment, a108P5H8 coding sequence is subcloned into the retroviral vectorpSRαMSVtkneo and used to infect various mammalian cell lines, such asNIH 3T3, TsuPr1, 293 and rat-1 in order to establish 108P5H8 expressingcell lines. Various other expression systems well known in the art canalso be employed. Expression constructs encoding a leader peptide joinedin frame to a 108P5H8 coding sequence can be used for the generation ofa secreted form of recombinant 108P5H8 protein.

[0175] As discussed herein, redundancy in the genetic code permitsvariation in 108P5H8 gene sequences. In particular, it is known in theart that specific host species often have specific codon preferences,and thus one can adapt the disclosed sequence as preferred for a desiredhost. For example, preferred analog codon sequences typically have rarecodons (i.e., codons having a usage frequency of less than about 20% inknown sequences of the desired host) replaced with higher frequencycodons. Codon preferences for a specific species are calculated, forexample, by utilizing codon usage tables available on the INTERNET suchas at URL www.dna.affrc.go.jp/˜nakamura/codon.html.

[0176] Additional sequence modifications are known to enhance proteinexpression in a cellular host. These include elimination of sequencesencoding spurious polyadenylation signals, exon/intron splice sitesignals, transposon-like repeats, and/or other such well-characterizedsequences that are deleterious to gene expression. The GC content of thesequence is adjusted to levels average for a given cellular host, ascalculated by reference to known genes expressed in the host cell. Wherepossible, the sequence is modified to avoid predicted hairpin secondarymRNA structures. Other useful modifications include the addition of atranslational initiation consensus sequence at the start of the openreading frame, as described in Kozak, Mol. Cell Biol., 9:5073-5080(1989). Skilled artisans understand that the general rule thateukaryotic ribosomes initiate translation exclusively at the 5′ proximalAUG codon is abrogated only under rare conditions (see, e.g., Kozak PNAS92(7): 2662-2666, (1995) and Kozak NAR 15(20): 8125-8148 (1987)).

[0177] III.) 108P5H8-Related Proteins

[0178] Another aspect of the present invention provides 108P5H8-relatedproteins. Specific embodiments of 108P5H8 proteins comprise apolypeptide having all or part of the amino acid sequence of human108P5H8 as shown in FIG. 2 or FIG. 3. Alternatively, embodiments of108P5H8 proteins comprise variant, homolog or analog polypeptides thathave alterations in the amino acid sequence of 108P5H8 shown in FIG. 2or FIG. 3.

[0179] In general, naturally occurring allelic variants of human 108P5H8share a high degree of structural identity and homology (e.g., 90% ormore homology). Typically, allelic variants of a 108P5H8 protein containconservative amino acid substitutions within the 108P5H8 sequencesdescribed herein or contain a substitution of an amino acid from acorresponding position in a homologue of 108P5H8. One class of 108P5H8allelic variants are proteins that share a high degree of homology withat least a small region of a particular 108P5H8 amino acid sequence, butfurther contain a radical departure from the sequence, such as anon-conservative substitution, truncation, insertion or frame shift. Incomparisons of protein sequences, the terms, similarity, identity, andhomology each have a distinct meaning as appreciated in the field ofgenetics. Moreover, orthology and paralogy can be important conceptsdescribing the relationship of members of a given protein family in oneorganism to the members of the same family in other organisms.

[0180] Amino acid abbreviations are provided in Table II. Conservativeamino acid substitutions can frequently be made in a protein withoutaltering either the conformation or the function of the protein.Proteins of the invention can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15 conservative substitutions. Such changes includesubstituting any of isoleucine (I), valine (V), and leucine (L) for anyother of these hydrophobic amino acids; aspartic acid (D) for glutamicacid (E) and vice versa; glutamine (Q) for asparagine (N) and viceversa; and serine (S) for threonine (T) and vice versa. Othersubstitutions can also be considered conservative, depending on theenvironment of the particular amino acid and its role in thethree-dimensional structure of the protein. For example, glycine (G) andalanine (A) can frequently be interchangeable, as can alanine (A) andvaline (V). Methionine (M), which is relatively hydrophobic, canfrequently be interchanged with leucine and isoleucine, and sometimeswith valine. Lysine (K) and arginine (R) are frequently-interchangeablein locations in which the significant feature of the amino acid residueis its charge and the differing pK's of these two amino acid residuesare not significant. Still other changes can be considered“conservative” in particular environments (see, e.g. Table III herein;pages 13-15 “Biochemistry” 2^(nd) ED. Lubert Stryer ed (StanfordUniversity); Henikoff et al. PNAS 1992 Vol 89 10915-10919; Lei et al., JBiol Chem May 19, 1995; 270(20):11882-6).

[0181] Embodiments of the invention disclosed herein include a widevariety of art-accepted variants or analogs of 108P5H8 proteins such aspolypeptides having amino acid insertions, deletions and substitutions.108P5H8 variants can be made using methods known in the art such assite-directed mutagenesis, alanine scanning, and PCR mutagenesis.Site-directed mutagenesis (Carter et al., Nucl. Acids Res., 13:4331(1986); Zoller et al., Nucl. Acids Res., 10:6487 (1987)), cassettemutagenesis (Wells et al., Gene, 34:315 (1985)), restriction selectionmutagenesis (Wells et al., Philos. Trans. R. Soc. London SerA, 317:415(1986)) or other known techniques can be performed on the cloned DNA toproduce the 108P5H8 variant DNA.

[0182] Scanning amino acid analysis can also be employed to identify oneor more amino acids along a contiguous sequence that is involved in aspecific biological activity such as a protein-protein interaction.Among the preferred scanning amino acids are relatively small, neutralamino acids. Such amino acids include alanine, glycine, serine, andcysteine. Alanine is typically a preferred scanning amino acid amongthis group because it eliminates the side-chain beyond the beta-carbonand is less likely to alter the main-chain conformation of the variant.Alanine is also typically preferred because it is the most common aminoacid. Further, it is frequently found in both buried and exposedpositions (Creighton, The Proteins, (W. H. Freeman & Co., N.Y.);Chothia, J. Mol. Biol., 150:1 (1976)). If alanine substitution does notyield adequate amounts of variant, an isosteric amino acid can be used.

[0183] As defined herein, 108P5H8 variants, analogs or homologs, havethe distinguishing attribute of having at least one epitope that is“cross reactive” with a 108P5H8 protein having an amino acid sequence ofFIG. 3. As used in this sentence, “cross reactive” means that anantibody or T cell that specifically binds to an 108P5H8 variant alsospecifically binds to a 108P5H8 protein having an amino acid sequenceset forth in FIG. 3. A polypeptide ceases to be a variant of a proteinshown in FIG. 3, when it no longer contains any epitope capable of beingrecognized by an antibody or T cell that specifically binds to thestarting 108P5H8 protein. Those skilled in the art understand thatantibodies that recognize proteins bind to epitopes of varying size, anda grouping of the order of about four or five amino acids, contiguous ornot, is regarded as a typical number of amino acids in a minimalepitope. See, e.g., Nair et al., J. Immunol 2000 165(12): 6949-6955;Hebbes et al., Mol Immunol (1989) 26(9):865-73; Schwartz et al, JImmunol (1985) 135(4):2598-608.

[0184] Other classes of 108P5H8-related protein variants share 70%, 75%,80%, 85% or 90% or more similarity with an amino acid sequence of FIG.3, or a fragment thereof. Another specific class of 108P5H8 proteinvariants or analogs comprise one or more of the 108P5H8 biologicalmotifs described herein or presently known in the art. Thus, encompassedby the present invention are analogs of 108P5H8 fragments (nucleic oramino acid) that have altered functional (e.g. immunogenic) propertiesrelative to the starting fragment. It is to be appreciated that motifsnow or which become part of the art are to be applied to the nucleic oramino acid sequences of FIG. 2 or FIG. 3.

[0185] As discussed herein, embodiments of the claimed invention includepolypeptides containing less than the full amino acid sequence of a108P5H8 protein shown in FIG. 2 or FIG. 3. For example, representativeembodiments of the invention comprise peptides/proteins having any 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more contiguous amino acids of a108P5H8 protein shown in FIG. 2 or FIG. 3.

[0186] Moreover, representative embodiments of the invention disclosedherein include polypeptides consisting of about amino acid 1 to aboutamino acid 10 of a 108P5H8 protein shown in FIG. 2 or FIG. 3,polypeptides consisting of about amino acid 10 to about amino acid 20 ofa 108P5H8 protein shown in FIG. 2 or FIG. 3, polypeptides consisting ofabout amino acid 20 to about amino acid 30 of a 108P5H8 protein shown inFIG. 2 or FIG. 3, polypeptides consisting of about amino acid 30 toabout amino acid 40 of a 108P5H8 protein shown in FIG. 2 or FIG. 3,polypeptides consisting of about amino acid 40 to about amino acid 50 ofa 108P5H8 protein shown in FIG. 2 or FIG. 3, polypeptides consisting ofabout amino acid 50 to about amino acid 60 of a 108P5H8 protein shown inFIG. 2 or FIG. 3, polypeptides consisting of about amino acid 60 toabout amino acid 70 of a 108P5H8 protein shown in FIG. 2 or FIG. 3,polypeptides consisting of about amino acid 70 to about amino acid 80 ofa 108P5H8 protein shown in FIG. 2 or FIG. 3, polypeptides consisting ofabout amino acid 80 to about amino acid 90 of a 108P5H8 protein shown inFIG. 2 or FIG. 3, polypeptides consisting of about amino acid 90 toabout amino acid 100 of a 108P5H8 protein shown in FIG. 2 or FIG. 3,etc. throughout the entirety of a 108P5H8 amino acid sequence. Moreover,polypeptides consisting of about amino acid 1 (or 20 or 30 or 40 etc.)to about amino acid 20, (or 130, or 140 or 150 etc.) of a 108P5H8protein shown in FIG. 2 or FIG. 3 are embodiments of the invention. Itis to be appreciated that the starting and stopping positions in thisparagraph refer to the specified position as well as that position plusor minus 5 residues.

[0187] 108P5H8-related proteins are generated using standard peptidesynthesis technology or using chemical cleavage methods well known inthe art. Alternatively, recombinant methods can be used to generatenucleic acid molecules that encode a 108P5H8-related protein. In oneembodiment, nucleic acid molecules provide a means to generate definedfragments of a 108P5H8 protein (or variants, homologs or analogsthereof).

[0188] III.A.) Motif-Bearing Protein Embodiments

[0189] Additional illustrative embodiments of the invention disclosedherein include 108P5H8 polypeptides comprising the amino acid residuesof one or more of the biological motifs contained within a 108P5H8polypeptide sequence set forth in FIG. 2 or FIG. 3. Various motifs areknown in the art, and a protein can be evaluated for the presence ofsuch motifs by a number of publicly available Internet sites (see, e.g.,URL addresses: pfam.wustl.edu/;http://searchlauncher.bcm.tmc.edu/seq-search/struc-predict.html;psort.ims.u-tokyo.acjp/; www.cbs.dtu.dk/;www.ebi.ac.uk/interpro/scan.html; www.expasy.ch/tools/scnpsit1.html;Epimatrix™ and Epimer™, Brown University,www.brown.edu/Research/TB-HIV_Lab/epimatrix/epimatrix.html; and BIMAS,bimas.dcrt.nih.gov/.).

[0190] Motif bearing subsequences of all 108P5H8 variant proteins areset forth and identified in Table XIX.

[0191] Table XX sets forth several frequently occurring motifs based onpfam searches (see URL address pfam.wustl.edu/). The columns of Table XXlist (1) motif name abbreviation, (2) percent identity found amongst thedifferent member of the motif family, (3) motif name or description and(4) most common function; location information is included if the motifis relevant for location.

[0192] Polypeptides comprising one or more of the 108P5H8 motifsdiscussed above are useful in elucidating the specific characteristicsof a malignant phenotype in view of the observation that the 108P5H8motifs discussed above are associated with growth dysregulation andbecause 108P5H8 is overexpressed in certain cancers (See, e.g., TableI). Casein kinase II, cAMP and camp-dependent protein kinase, andProtein Kinase C, for example, are enzymes known to be associated withthe development of the malignant phenotype (see e.g. Chen et al., LabInvest., 78(2): 165-174 (1998); Gaiddon et al., Endocrinology 136(10):4331-4338 (1995); Hall et al., Nucleic Acids Research 24(6): 1119-1126(1996); Peterziel et al., Oncogene 18(46): 6322-6329 (1999) and O'Brian,Oncol. Rep. 5(2): 305-309 (1998)). Moreover, both glycosylation andmyristoylation are protein modifications also associated with cancer andcancer progression (see e.g. Dennis et al., Biochem. Biophys. Acta1473(1):21-34 (1999); Raju et al., Exp. Cell Res. 235(1): 145-154(1997)). Amidation is another protein modification also associated withcancer and cancer progression (see e.g. Treston et al., J. Natl. CancerInst. Monogr. (13): 169-175 (1992)).

[0193] In another embodiment, proteins of the invention comprise one ormore of the immunoreactive epitopes identified in accordance withart-accepted methods, such as the peptides set forth in Tables V-XVIII,XXII, and XXIII. CTL epitopes can be determined using specificalgorithms to identify peptides within an 108P5H8 protein that arecapable of optimally binding to specified HLA alleles (e.g., Table IV;Epimatrix™ and Epimer™, Brown University, URLwww.brown.edu/Research/TB-HIV_Lab/epimatrix/epimatrix.html; and BIMAS,URL bimas.dcrt.nih.gov/.) Moreover, processes for identifying peptidesthat have sufficient binding affinity for HLA molecules and which arecorrelated with being immunogenic epitopes, are well known in the art,and are carried out without undue experimentation. In addition,processes for identifying peptides that are immunogenic epitopes, arewell known in the art, and are carried out without undue experimentationeither in vitro or in vivo.

[0194] Also known in the art are principles for creating analogs of suchepitopes in order to modulate immunogenicity. For example, one beginswith an epitope that bears a CTL or HTL motif (see, e.g., the HLA ClassI and HLA Class II motifs/supermotifs of Table IV). The epitope isanaloged by substituting out an amino acid at one of the specifiedpositions, and replacing it with another amino acid specified for thatposition. For example, one can substitute out a deleterious residue infavor of any other residue, such as a preferred residue as defined inTable IV; substitute a less-preferred residue with a preferred residueas defined in Table IV; or substitute an originally-occurring preferredresidue with another preferred residue as defined in Table IV.Substitutions can occur at primary anchor positions or at otherpositions in a peptide; see, e.g., Table IV.

[0195] A variety of references reflect the art regarding theidentification and generation of epitopes in a protein of interest aswell as analogs thereof. See, for example, WO 9733602 to Chesnut et al.;Sette, Immunogenetics 1999 50(3-4): 201-212; Sette et al., J. Immunol.2001 166(2): 1389-1397; Sidney et al., Hum. Immunol. 1997 58(1): 12-20;Kondo et al., Immunogenetics 1997 45(4): 249-258; Sidney et al., J.Immunol. 1996 157(8): 3480-90; and Falk et al., Nature 351: 290-6(1991); Hunt et al., Science 255:1261-3 (1992); Parker et al., J.Immunol. 149:3580-7 (1992); Parker et al., J. Immunol. 152:163-75(1994); Kast et al., 1994 152(8): 3904-12; Borras-Cuesta et al., Hum.Immunol. 2000 61(3): 266-278; Alexander et al., J. Immunol. 2000 164(3);164(3): 1625-1633; Alexander et al., PMID: 7895164, UI: 95202582;O'Sullivan et al., J. Immunol. 1991 147(8): 2663-2669; Alexander et al.,Immunity 1994 1(9): 751-761 and Alexander et al., Immunol. Res. 199818(2): 79-92.

[0196] Related embodiments of the inventions include polypeptidescomprising combinations of the different motifs set forth in Table XIX,and/or, one or more of the predicted CTL epitopes of Tables V-XVIII,Table XXII, Table XXIII, and/or, one or more of the T cell bindingmotifs known in the art. Preferred embodiments contain no insertions,deletions or substitutions either within the motifs or the interveningsequences of the polypeptides. In addition, embodiments which include anumber of either N-terminal and/or C-terminal amino acid residues oneither side of these motifs may be desirable (to, for example, include agreater portion of the polypeptide architecture in which the motif islocated). Typically the number of N-terminal and/or C-terminal aminoacid residues on either side of a motif is between about 1 to about 100amino acid residues, preferably 5 to about 50 amino acid residues.

[0197] 108P5H8-related proteins are embodied in many forms, preferablyin isolated form. A purified 108P5H8 protein molecule will besubstantially free of other proteins or molecules that impair thebinding of 108P5H8 to antibody, T cell or other ligand. The nature anddegree of isolation and purification will depend on the intended use.Embodiments of a 108P5H8-related proteins include purified108P5H8-related proteins and functional, soluble 108P5H8-relatedproteins. In one embodiment, a functional, soluble 108P5H8 protein orfragment thereof retains the ability to be bound by antibody, T cell orother ligand.

[0198] The invention also provides 108P5H8 proteins comprisingbiologically active fragments of a 108P5H8 amino acid sequence shown inFIG. 2 or FIG. 3. Such proteins exhibit properties of the starting108P5H8 protein, such as the ability to elicit the generation ofantibodies that specifically bind an epitope associated with thestarting 108P5H8 protein; to be bound by such antibodies; to elicit theactivation of HTL or CTL; and/or, to be recognized by HTL or CTL thatalso specifically bind to the starting protein.

[0199] 108P5H8-related polypeptides that contain particularlyinteresting structures can be predicted and/or identified using variousanalytical techniques well known in the art, including, for example, themethods of Chou-Fasman, Garnier-Robson, Kyte-Doolittle, Eisenberg,Karplus-Schultz or Jameson-Wolf analysis, or on the basis ofimmunogenicity. Fragments that contain such structures are particularlyuseful in generating subunit-specific anti-108P5H8 antibodies, or Tcells or in identifying cellular factors that bind to 108P5H8.

[0200] CTL epitopes can be determined using specific algorithms toidentify peptides within an 108P5H8 protein that are capable ofoptimally binding to specified HLA alleles (e.g., by using the SYFPEITHIsite at World Wide Web URL syfpeithi.bmi-heidelberg.com/; the listingsin Table IV(A)-(E); Epimatrix™ and Epimer™, Brown University, URL(www.brown.edu/Research/TB-HIV_Lab/epimatrix/epimatrix.html); and BIMAS,URL bimas.dcrt.nih.gov/). Illustrating this, peptide epitopes from108P5H8 that are presented in the context of human MHC class I moleculesHLA-A1, A2, A3, A11, A24, B7 and B35 were predicted (Tables V-XVIII,XXII, and XXIII). Specifically, the complete amino acid sequence of the108P5H8 protein and relevant portions of other variants, i.e., for HLAClass I predictions 9 flanking redisues on either side of a pointmutation, and for HLA Class II predictions 14 flanking residues oneither side of a point mutation, were entered into the HLA Peptide MotifSearch algorithm found in the Bioinformatics and Molecular AnalysisSection (BIMAS) web site listed above; for HLA Class II the siteSYFPEITHI at URL syfpeithi.bmi-heidelberg.com/ was used.

[0201] The HLA peptide motif search algorithm was developed by Dr. KenParker based on binding of specific peptide sequences in the groove ofHLA Class I molecules, in particular HLA-A2 (see, e.g., Falk et al.,Nature 351: 290-6 (1991); Hunt et al., Science 255:1261-3 (1992); Parkeret al., J. Immunol. 149:3580-7(1992); Parker et al., J. Immunol.152:163-75 (1994)). This algorithm allows location and ranking of 8-mer, 9 -mer, and 10 -mer peptides from a complete protein sequence forpredicted binding to HLA-A2 as well as numerous other HLA Class Imolecules. Many HLA class I binding peptides are 8-, 9-, 10 or 11 -mers.For example, for class I HLA-A2, the epitopes preferably contain aleucine (L) or methionine (M) at position 2 and a valine (V) or leucine(L) at the C-terminus (see, e.g., Parker et al., J. Immunol. 149:3580-7(1992)). Selected results of 108P5H8 predicted binding peptides areshown in Tables V-XVIII, XXII, and XXIII herein. In Tables V-XVIII, thetop 50 ranking candidates, 9 -mers and 10 -mers, for each family memberare shown along with their location, the amino acid sequence of eachspecific peptide, and an estimated binding score. The binding scorecorresponds to the estimated half time of dissociation of complexescontaining the peptide at 37° C. at pH 6.5. Peptides with the highestbinding score are predicted to be the most tightly bound to HLA Class Ion the cell surface for the greatest period of time and thus representthe best immunogenic targets for T-cell recognition.

[0202] Actual binding of peptides to an HLA allele can be evaluated bystabilization of HLA expression on the antigen-processing defective cellline T2 (see, e.g., Xue et al., Prostate 30:73-8 (1997) and Peshwa etal., Prostate 36:129-38 (1998)). Immunogenicity of specific peptides canbe evaluated in vitro by stimulation of CD8+ cytotoxic T lymphocytes(CTL) in the presence of antigen presenting cells such as dendriticcells.

[0203] It is to be appreciated that every epitope predicted by the BIMASsite, Epimer™ and Epimatrix™ sites, or specified by the HLA class I orclass II motifs available in the art or which become part of the artsuch as set forth in Table IV (or determined using World Wide Web siteURL syfpeithi.bmi-heidelberg.com/, or BIMAS, bimas.dcrt.nih.gov/) are tobe “applied” to a 108P5H8 protein in accordance with the invention. Asused in this context “applied” means that a 108P5H8 protein isevaluated, e.g., visually or by computer-based patterns finding methods,as appreciated by those of skill in the relevant art. Every subsequenceof a 108P5H8 protein of 8, 9, 10, or 11 amino acid residues that bearsan HLA Class I motif, or a subsequence of 9 or more amino acid residuesthat bear an HLA Class II motif are within the scope of the invention.

[0204] III.B.) Expression of 108P5H8-Related Proteins

[0205] In an embodiment described in the examples that follow, 108P5H8can be conveniently expressed in cells (such as 293T cells) transfectedwith a commercially available expression vector such as a CMV-drivenexpression vector encoding 108P5H8 with a C-terminal 6×His and MYC tag(pcDNA3.1/mycHIS, Invitrogen or Tag5, GenHunter Corporation, NashvilleTenn.). The Tag5 vector provides an IgGK secretion signal that can beused to facilitate the production of a secreted 108P5H8 protein intransfected cells. The secreted HIS-tagged 108P5H8 in the culture mediacan be purified, e.g., using a nickel column using standard techniques.

[0206] III.C.) Modifications of 108P5H8-Related Proteins

[0207] Modifications of 108P5H8-related proteins such as covalentmodifications are included within the scope of this invention. One typeof covalent modification includes reacting targeted amino acid residuesof a 108P5H8 polypeptide with an organic derivatizing agent that iscapable of reacting with selected side chains or the N- or C-terminalresidues of a 108P5H8 protein. Another type of covalent modification ofa 108P5H8 polypeptide included within the scope of this inventioncomprises altering the native glycosylation pattern of a protein of theinvention. Another type of covalent modification of 108P5H8 compriseslinking a 108P5H8 polypeptide to one of a variety of nonproteinaceouspolymers, e.g., polyethylene glycol (PEG), polypropylene glycol, orpolyoxyalkylenes, in the manner set forth in U.S. Pat. Nos. 4,640,835;4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337.

[0208] The 108P5H8-related proteins of the present invention can also bemodified to form a chimeric molecule comprising 108P5H8 fused toanother, heterologous polypeptide or amino acid sequence. Such achimeric molecule can be synthesized chemically or recombinantly. Achimeric molecule can have a protein of the invention fused to anothertumor-associated antigen or fragment thereof. Alternatively, a proteinin accordance with the invention can comprise a fusion of fragments of a108P5H8 sequence (amino or nucleic acid) such that a molecule is createdthat is not, through its length, directly homologous to the amino ornucleic acid sequences shown in FIG. 2 or FIG. 3. Such a chimericmolecule can comprise multiples of the same subsequence of 108P5H8. Achimeric molecule can comprise a fusion of a 108P5H8-related proteinwith a polyhistidine epitope tag, which provides an epitope to whichimmobilized nickel can selectively bind, with cytokines or with growthfactors. The epitope tag is generally placed at the amino- orcarboxyl-terminus of a 108P5H8 protein. In an alternative embodiment,the chimeric molecule can comprise a fusion of a 108P5H8-related proteinwith an immunoglobulin or a particular region of an immunoglobulin. Fora bivalent form of the chimeric molecule (also referred to as an“immunoadhesin”), such a fusion could be to the Fe region of an IgGmolecule. The Ig fusions preferably include the substitution of asoluble (transmembrane domain deleted or inactivated) form of a 108P5H8polypeptide in place of at least one variable region within an Igmolecule. In a preferred embodiment, the immunoglobulin fusion includesthe hinge, CH2 and CH3, or the hinge, CHI, CH2 and CH3 regions of anIgGI molecule. For the production of immunoglobulin fusions see, e.g.,U.S. Pat. No. 5,428,130 issued Jun. 27, 1995.

[0209] III.D.) Uses of 108P5H8-Related Proteins

[0210] The proteins of the invention have a number of different specificuses. As 108P5H8 is highly expressed in prostate and other cancers,108P5H8-related proteins are used in methods that assess the status of108P5H8 gene products in normal versus cancerous tissues, therebyelucidating the malignant phenotype. Typically, polypeptides fromspecific regions of a 108P5H8 protein are used to assess the presence ofperturbations (such as deletions, insertions, point mutations etc.) inthose regions (such as regions containing one or more motifs). Exemplaryassays utilize antibodies or T cells targeting 108P5H8-related proteinscomprising the amino acid residues of one or more of the biologicalmotifs contained within a 108P5H8 polypeptide sequence in order toevaluate the characteristics of this region in normal versus canceroustissues or to elicit an immune response to the epitope. Alternatively,108P5H8-related proteins that contain the amino acid residues of one ormore of the biological motifs in a 108P5H8 protein are used to screenfor factors that interact with that region of 108P5H8.

[0211] 108P5H8 protein fragments/subsequences are particularly useful ingenerating and characterizing domain-specific antibodies (e.g.,antibodies recognizing an extracellular or intracellular epitope of an108P5H8 protein), for identifying agents or cellular factors that bindto 108P5H8 or a particular structural domain thereof, and in varioustherapeutic and diagnostic contexts, including but not limited todiagnostic assays, cancer vaccines and methods of preparing suchvaccines.

[0212] Proteins encoded by the 108P5H8 genes, or by analogs, homologs orfragments thereof, have a variety of uses, including but not limited togenerating antibodies and in methods for identifying ligands and otheragents and cellular constituents that bind to an 108P5H8 gene product.Antibodies raised against an 108P5H8 protein or fragment thereof areuseful in diagnostic and prognostic assays, and imaging methodologies inthe management of human cancers characterized by expression of 108P5H8protein, such as those listed in Table I. Such antibodies can beexpressed intracellularly and used in methods of treating patients withsuch cancers. 108P5H8-related nucleic acids or proteins are also used ingenerating HTL or CTL responses.

[0213] Various immunological assays useful for the detection of 108P5H8proteins are used, including but not limited to various types ofradioimmunoassay, enzyme-linked immunosorbent assays (ELISA),enzyme-linked immunofluorescent assays (ELIFA), immunocytochemicalmethods, and the like. Antibodies can be labeled and used asimmunological imaging reagents capable of detecting 108P5H8-expressingcells (e.g., in radioscintigraphic imaging methods). 108P5H8 proteinsare also particularly useful in generating cancer vaccines, as furtherdescribed herein.

[0214] IV.) 108P5H8 Antibodies

[0215] Another aspect of the invention provides antibodies that bind to108P5H8-related proteins. Preferred antibodies specifically bind to a108P5H8-related protein and do not bind (or bind weakly) to peptides orproteins that are not 108P5H8-related proteins. For example, antibodiesthat bind 108P5H8 can bind 108P5H8-related proteins such as the homologsor analogs thereof.

[0216] 108P5H8 antibodies of the invention are particularly useful incancer (see, e.g., Table I) diagnostic and prognostic assays, andimaging methodologies. Similarly, such antibodies are useful in thetreatment, diagnosis, and/or prognosis of other cancers, to the extent108P5H8 is also expressed or overexpressed in these other cancers.Moreover, intracellularly expressed antibodies (e.g., single chainantibodies) are therapeutically useful in treating cancers in which theexpression of 108P5H8 is involved, such as advanced or metastaticprostate cancers.

[0217] The invention also provides various immunological assays usefulfor the detection and quantification of 108P5H8 and mutant108P5H8-related proteins. Such assays can comprise one or more 108P5H8antibodies capable of recognizing and binding a 108P5H8-related protein,as appropriate. These assays are performed within various immunologicalassay formats well known in the art, including but not limited tovarious types of radioimmunoassays, enzyme-linked immunosorbent assays(ELISA), enzyme-linked immunofluorescent assays (ELIFA), and the like.

[0218] Immunological non-antibody assays of the invention also compriseT cell immunogenicity assays (inhibitory or stimulatory) as well asmajor histocompatibility complex (MHC) binding assays.

[0219] In addition, immunological imaging methods capable of detectingprostate cancer and other cancers expressing 108P5H8 are also providedby the invention, including but not limited to radioscintigraphicimaging methods using labeled 108P5H8 antibodies. Such assays areclinically useful in the detection, monitoring, and prognosis of 108P5H8expressing cancers such as prostate cancer.

[0220] 108P5H8 antibodies are also used in methods for purifying a108P5H8-related protein and for isolating 108P5H8 homologues and relatedmolecules. For example, a method of purifying a 108P5H8-related proteincomprises incubating an 108P5H8 antibody, which has been coupled to asolid matrix, with a lysate or other solution containing a108P5H8-related protein under conditions that permit the 108P5H8antibody to bind to the 108P5H8-related protein; washing the solidmatrix to eliminate impurities; and eluting the 108P5H8-related proteinfrom the coupled antibody. Other uses of 108P5H8 antibodies inaccordance with the invention include generating anti-idiotypicantibodies that mimic a 108P5H8 protein.

[0221] Various methods for the preparation of antibodies are well knownin the art. For example, antibodies can be prepared by immunizing asuitable mammalian host using a 108P5H8-related protein, peptide, orfragment, in isolated or immunoconjugated form (Antibodies: A LaboratoryManual, CSH Press, Eds., Harlow, and Lane (1988); Harlow, Antibodies,Cold Spring Harbor Press, NY (1989)). In addition, fusion proteins of108P5H8 can also be used, such as a 108P5H8 GST-fusion protein. In aparticular embodiment, a GST fusion protein comprising all or most ofthe amino acid sequence of FIG. 2 or FIG. 3 is produced, then used as animmunogen to generate appropriate antibodies. In another embodiment, a108P5H8-related protein is synthesized and used as an immunogen.

[0222] In addition, naked DNA immunization techniques known in the artare used (with or without purified 108P5H8-related protein or 108P5H8expressing cells) to generate an immune response to the encodedimmunogen (for review, see Donnelly et al., 1997, Ann. Rev. Immunol. 15:617-648).

[0223] The amino acid sequence of a 108P5H8 protein as shown in FIG. 2or FIG. 3 can be analyzed to select specific regions of the 108P5H8protein for generating antibodies. For example, hydrophobicity andhydrophilicity analyses of a 108P5H8 amino acid sequence are used toidentify hydrophilic regions in the 108P5H8 structure. Regions of a108P5H8 protein that show immunogenic structure, as well as otherregions and domains, can readily be identified using various othermethods known in the art, such as Chou-Fasman, Garnier-Robson,Kyte-Doolittle, Eisenberg, Karplus-Schultz or Jameson-Wolf analysis.Thus, each region identified by any of these programs or methods iswithin the scope of the present invention. Methods for the generation of108P5H8 antibodies are further illustrated by way of the examplesprovided herein. Methods for preparing a protein or polypeptide for useas an immunogen are well known in the art. Also well known in the artare methods for preparing immunogenic conjugates of a protein with acarrier, such as BSA, KLH or other carrier protein. In somecircumstances, direct conjugation using, for example, carbodiimidereagents are used; in other instances lining reagents such as thosesupplied by Pierce Chemical Co., Rockford, Ill., are effective.Administration of a 108P5H8 immunogen is often conducted by injectionover a suitable time period and with use of a suitable adjuvant, as isunderstood in the art. During the immunization schedule, titers ofantibodies can be taken to determine adequacy of antibody formation.

[0224] 108P5H8 monoclonal antibodies can be produced by various meanswell known in the art. For example, immortalized cell lines that secretea desired monoclonal antibody are prepared using the standard hybridomatechnology of Kohler and Milstein or modifications that immortalizeantibody-producing B cells, as is generally known. Immortalized celllines that secrete the desired antibodies are screened by immunoassay inwhich the antigen is a 108P5H8-related protein. When the appropriateimmortalized cell culture is identified, the cells can be expanded andantibodies produced either from in vitro cultures or from ascites fluid.

[0225] The antibodies or fragments of the invention can also beproduced, by recombinant means. Regions that bind specifically to thedesired regions of a 108P5H8 protein can also be produced in the contextof chimeric or complementarity determining region (CDR) graftedantibodies of multiple species origin. Humanized or human 108P5H8antibodies can also be produced, and are preferred for use intherapeutic contexts. Methods for humanizing murine and other non-humanantibodies, by substituting one or more of the non-human antibody CDRsfor corresponding human antibody sequences, are well known (see forexample, Jones et al., 1986, Nature 321: 522-525; Riechmann et al.,1988, Nature 332: 323-327; Verhoeyen et al., 1988, Science 239:1534-1536). See also, Carter et al., 1993, Proc. Natl. Acad. Sci. USA89: 4285 and Sims et al., 1993, J. Immunol. 151: 2296.

[0226] Methods for producing fully human monoclonal antibodies includephage display and transgenic methods (for review, see Vaughan et al.,1998, Nature Biotechnology 16: 535-539). Fully human 108P5H8 monoclonalantibodies can be generated using cloning technologies employing largehuman Ig gene combinatorial libraries (i.e., phage display) (Griffithsand Hoogenboom, Building an in vitro immune system: human antibodiesfrom phage display libraries. In: Protein Engineering of AntibodyMolecules for Prophylactic and Therapeutic Applications in Man, Clark,M. (Ed.), Nottingham Academic, pp 45-64 (1993); Burton and Barbas, HumanAntibodies from combinatorial libraries. Id., pp 65-82). Fully human108P5H8 monoclonal antibodies can also be produced using transgenic miceengineered to contain human immunoglobulin gene loci as described in PCTPatent Application WO98/24893, Kucherlapati and Jakobovits et al.,published Dec. 3, 1997 (see also, Jakobovits, 1998, Exp. Opin. Invest.Drugs 7(4): 607-614; U.S. Pat. Nos. 6,162,963 issued Dec. 19, 2000;6,150,584 issued Nov. 12, 2000; and, 6,114598 issued Sep. 5, 2000). Thismethod avoids the in vitro manipulation required with phage displaytechnology and efficiently produces high affinity authentic humanantibodies.

[0227] Reactivity of 108P5H8 antibodies with an 108P5H8-related proteincan be established by a number of well known means, including Westernblot, immunoprecipitation, ELISA, and FACS analyses using, asappropriate, 108P5H8-related proteins, 108P5H8-expressing cells orextracts thereof. A 108P5H8 antibody or fragment thereof can be labeledwith a detectable marker or conjugated to a second molecule. Suitabledetectable markers include, but are not limited to, a radioisotope, afluorescent compound, a bioluminescent compound, chemiluminescentcompound, a metal chelator or an enzyme. Further, bi-specific antibodiesspecific for two or more 108P5H8 epitopes are generated using methodsgenerally known in the art. Homodimeric antibodies can also be generatedby cross-linking techniques known in the art (e.g., Wolff et al., CancerRes. 53: 2560-2565).

[0228] V.) 108P5H8 Cellular Immune Responses

[0229] The mechanism by which T cells recognize antigens has beendelineated. Efficacious peptide epitope vaccine compositions of theinvention induce a therapeutic or prophylactic immune responses in verybroad segments of the world-wide population. For an understanding of thevalue and efficacy of compositions of the invention that induce cellularimmune responses, a brief review of immunology-related technology isprovided.

[0230] A complex of an HLA molecule and a peptidic antigen acts as theligand recognized by HLA-restricted T cells (Buus, S. et al., Cell47:1071, 1986; Babbitt, B. P. et al., Nature 317:359, 1985; Townsend, A.and Bodmer, H., Annu. Rev. Immunol. 7:601, 1989; Germain, R. N., Annu.Rev. Immunol. 11:403, 1993). Through the study of single amino acidsubstituted antigen analogs and the sequencing of endogenously bound,naturally processed peptides, critical residues that correspond tomotifs required for specific binding to HLA antigen molecules have beenidentified and are set forth in Table IV (see also, e.g., Southwood, etal., J. Immunol. 160:3363, 1998; Rammensee, et al., Immunogenetics41:178, 1995; Rammensee et al., SYFPEITHI, access via World Wide Web atURL syfpeithi.bmi-heidelberg.com/; Sette, A. and Sidney, J. Curr. Opin.Immunol. 10:478, 1998; Engelhard, V. H., Curr. Opin. Immunol. 6:13,1994; Sette, A. and Grey, H. M., Curr. Opin. Immunol. 4:79, 1992;Sinigaglia, F. and Hammer, J. Curr. Biol. 6:52, 1994; Ruppert et al.,Cell 74:929-937, 1993; Kondo et al., J. Immunol. 155:4307-4312, 1995;Sidney et al., J. Immunol. 157:3480-3490, 1996; Sidney et al., HumanImmunol. 45:79-93, 1996; Sette, A. and Sidney, J. ImmunogeneticsNovember 1999; 50(3-4):201-12, Review).

[0231] Furthermore, x-ray crystallographic analyses of HLA-peptidecomplexes have revealed pockets within the peptide binding cleft/grooveof HLA molecules which accommodate, in an allele-specific mode, residuesborne by peptide ligands; these residues in turn determine the HLAbinding capacity of the peptides in which they are present. (See, e.g.,Madden, D. R. Annu. Rev. Immunol. 13:587, 1995; Smith, et al., Immunity4:203, 1996; Fremont et al., Immunity 8:305, 1998; Stern et al.,Structure 2:245, 1994; Jones, E. Y. Curr. Opin. Immunol. 9:75, 1997;Brown, J. H. et al., Nature 364:33, 1993; Guo, H. C. et al., Proc. Natl.Acad. Sci. USA 90:8053, 1993; Guo, H. C. et al., Nature 360:364, 1992;Silver, M. L. et al., Nature 360:367, 1992; Matsumura, M. et al.,Science 257:927, 1992; Madden et al., Cell 70:1035, 1992; Fremont, D. H.et al., Science 257:919, 1992; Saper, M. A., Bjorkman, P. J. and Wiley,D. C., J. Mol. Biol. 219:277, 1991.)

[0232] Accordingly, the definition of class I and class IIallele-specific HLA binding motifs, or class I or class II supermotifsallows identification of regions within a protein that are correlatedwith binding to particular HLA antigen(s).

[0233] Thus, by a process of HLA motif identification, candidates forepitope-based vaccines have been identified; such candidates can befurther evaluated by HLA-peptide binding assays to determine bindingaffinity and/or the time period of association of the epitope and itscorresponding HLA molecule. Additional confirmatory work can beperformed to select, amongst these vaccine candidates, epitopes withpreferred characteristics in terms of population coverage, and/orimmunogenicity.

[0234] Various strategies can be utilized to evaluate cellularimmunogenicity, including:

[0235] 1) Evaluation of primary T cell cultures from normal individuals(see, e.g., Wentworth, P. A. et al., Mol. Immunol. 32:603, 1995; Celis,E. et al., Proc. Natl. Acad. Sci. USA 91:2105, 1994; Tsai, V. et al., J.Immunol. 158-1796, 1997; Kawashima, I. et al., Human Immunol. 59:1,1998). This procedure involves the stimulation of peripheral bloodlymphocytes (PBL) from normal subjects with a test peptide in thepresence of antigen presenting cells in vitro over a period of severalweeks. T cells specific for the peptide become activated during thistime and are detected using, e.g., a lymphokine- or ⁵¹Cr-release assayinvolving peptide sensitized target cells.

[0236] 2) Immunization of HLA transgenic mice (see, e.g., Wentworth, P.A. et al., J. Immunol. 26:97, 1996; Wentworth, P. A. et al., Int.Immunol. 8:651, 1996; Alexander, J. et al., J. Immunol. 159:4753, 1997).For example, in such methods peptides in incomplete Freund's adjuvantare administered subcutaneously to HLA transgenic mice. Several weeksfollowing immunization, splenocytes are removed and cultured in vitro inthe presence of test peptide for approximately one week.Peptide-specific T cells are detected using, e.g., a ⁵¹Cr-release assayinvolving peptide sensitized target cells and target cells expressingendogenously generated antigen.

[0237] 3) Demonstration of recall T cell responses from immuneindividuals who have been either effectively vaccinated and/or fromchronically ill patients (see, e.g., Rehermann, B. et al., J. Exp. Med.181:1047, 1995; Doolan, D. L. et al., Immunity 7:97, 1997; Bertoni, R.et al., J. Clin. Invest. 100:503, 1997; Threlkeld, S. C. et al., J.Immunol. 159:1648, 1997; Diepolder, H. M. et al., J. Virol. 71:6011,1997). Accordingly, recall responses are detected by culturing PBL fromsubjects that have been exposed to the antigen due to disease and thushave generated an immune response “naturally”, or from patients who werevaccinated against the antigen. PBL from subjects are cultured in vitrofor 1-2 weeks in the presence of test peptide plus antigen presentingcells (APC) to allow activation of “memory” T cells, as compared to“naive” T cells. At the end of the culture period, T cell activity isdetected using assays including ⁵¹Cr release involvingpeptide-sensitized targets, T cell proliferation, or lymphokine release.

[0238] VI.) 108P5H8 Transgenic Animals

[0239] Nucleic acids that encode a 108P5H8-related protein can also beused to generate either transgenic animals or “knock out” animals that,in turn, are useful in the development and screening of therapeuticallyuseful reagents. In accordance with established techniques, cDNAencoding 108P5H8 can be used to clone genomic DNA that encodes 108P5H8.The cloned genomic sequences can then be used to generate transgenicanimals containing cells that express DNA that encode 108P5H8. Methodsfor generating transgenic animals, particularly animals such as mice orrats, have become conventional in the art and are described, forexample, in U.S. Pat. Nos. 4,736,866 issued Apr. 12, 1988, and 4,870,009issued Sep. 26, 1989. Typically, particular cells would be targeted for108P5H8 transgene incorporation with tissue-specific enhancers.

[0240] Transgenic animals that include a copy of a transgene encoding108P5H8 can be used to examine the effect of increased expression of DNAthat encodes 108P5H8. Such animals can be used as tester animals forreagents thought to confer protection from, for example, pathologicalconditions associated with its overexpression. In accordance with thisaspect of the invention, an animal is treated with a reagent and areduced incidence of a pathological condition, compared to untreatedanimals that bear the transgene, would indicate a potential therapeuticintervention for the pathological condition.

[0241] Alternatively, non-human homologues of 108P5H8 can be used toconstruct a 108P5H8 “knock out” animal that has a defective or alteredgene encoding 108P5H8 as a result of homologous recombination betweenthe endogenous gene encoding 108P5H8 and altered genomic DNA encoding108P5H8 introduced into an embryonic cell of the animal. For example,cDNA that encodes 108P5H8 can be used to clone genomic DNA encoding108P5H8 in accordance with established techniques. A portion of thegenomic DNA encoding 108P5H8 can be deleted or replaced with anothergene, such as a gene encoding a selectable marker that can be used tomonitor integration. Typically, several kilobases of unaltered flankingDNA (both at the 5′ and 3′ ends) are included in the vector (see, e.g.,Thomas and Capecchi, Cell, 51:503 (1987) for a description of homologousrecombination vectors). The vector is introduced into an embryonic stemcell line (e.g., by electroporation) and cells in which the introducedDNA has homologously recombined with the endogenous DNA are selected(see, e.g., Li et al., Cell, 69:915 (1992)). The selected cells are theninjected into a blastocyst of an animal (e.g., a mouse or rat) to formaggregation chimeras (see, e.g., Bradley, in Teratocarcinomas andEmbryonic Stem Cells: A Practical Approach, E. J. Robertson, ed. (IRL,Oxford, 1987), pp. 113-152). A chimeric embryo can then be implantedinto a suitable pseudopregnant female foster animal, and the embryobrought to term to create a “knock out” animal. Progeny harboring thehomologously recombined DNA in their germ cells can be identified bystandard techniques and used to breed animals in which all cells of theanimal contain the homologously recombined DNA. Knock out animals can becharacterized, for example, for their ability to defend against certainpathological conditions or for their development of pathologicalconditions due to absence of a 108P5H8 polypeptide.

[0242] VII.) Methods for the Detection of 108P5H8

[0243] Another aspect of the present invention relates to methods fordetecting 108P5H8 polynucleotides and 108P5H8-related proteins, as wellas methods for identifying a cell that expresses 108P5H8. The expressionprofile of 108P5H8 makes it a diagnostic marker for metastasizeddisease. Accordingly, the status of 108P5H8 gene products providesinformation useful for predicting a variety of factors includingsusceptibility to advanced stage disease, rate of progression, and/ortumor aggressiveness. As discussed in detail herein, the status of108P5H8 gene products in patient samples can be analyzed by a varietyprotocols that are well known in the art including immunohistochemicalanalysis, the variety of Northern blotting techniques including in situhybridization, RT-PCR analysis (for example on laser capturemicro-dissected samples), Western blot analysis and tissue arrayanalysis.

[0244] More particularly, the invention provides assays for thedetection of 108P5H8 polynucleotides in a biological sample, such asserum, bone, prostate, and other tissues, urine, semen, cellpreparations, and the like. Detectable 108P5H8 polynucleotides include,for example, a 108P5H8 gene or fragment thereof, 108P5H8 mRNA,alternative splice variant 108P5H8 mRNAs, and recombinant DNA or RNAmolecules that contain a 108P5H8 polynucleotide. A number of methods foramplifying and/or detecting the presence of 108P5H8 polynucleotides arewell known in the art and can be employed in the practice of this aspectof the invention.

[0245] In one embodiment, a method for detecting an 108P5H8 mRNA in abiological sample comprises producing cDNA from the sample by reversetranscription using at least one primer; amplifying the cDNA so producedusing an 108P5H8 polynucleotides as sense and antisense primers toamplify 108P5H8 cDNAs therein; and detecting the presence of theamplified 108P5H8 cDNA. Optionally, the sequence of the amplified108P5H8 cDNA can be determined.

[0246] In another embodiment, a method of detecting a 108P5H8 gene in abiological sample comprises first isolating genomic DNA from the sample;amplifying the isolated genomic DNA using 108P5H8 polynucleotides assense and antisense primers; and detecting the presence of the amplified108P5H8 gene. Any number of appropriate sense and antisense probecombinations can be designed from a 192P1E1B nucleotide sequence (see,e.g., FIG. 2) and used for this purpose.

[0247] The invention also provides assays for detecting the presence ofan 108P5H8 protein in a tissue or other biological sample such as serum,semen, bone, prostate, urine, cell preparations, and the like. Methodsfor detecting a 108P5H8-related protein are also well known and include,for example, immunoprecipitation, immunohistochemical analysis, Westernblot analysis, molecular binding assays, ELISA, ELIFA and the like. Forexample, a method of detecting the presence of a 108P5H8-related proteinin a biological sample comprises first contacting the sample with a108P5H8 antibody, a 108P5H8-reactive fragment thereof, or a recombinantprotein containing an antigen binding region of a 108P5H8 antibody; andthen detecting the binding of 108P5H8-related protein in the sample.

[0248] Methods for identifying a cell that expresses 108P5H8 are alsowithin the scope of the invention. In one embodiment, an assay foridentifying a cell that expresses a 108P5H8 gene comprises detecting thepresence of 108P5H8 mRNA in the cell. Methods for the detection ofparticular mRNAs in cells are well known and include, for example,hybridization assays using complementary DNA probes (such as in situhybridization using labeled 108P5H8 riboprobes, Northern blot andrelated techniques) and various nucleic acid amplification assays (suchas RT-PCR using complementary primers specific for 108P5H8, and otheramplification type detection methods, such as, for example, branchedDNA, SISBA, TMA and the like). Alternatively, an assay for identifying acell that expresses a 108P5H8 gene comprises detecting the presence of108P5H8-related protein in the cell or secreted by the cell. Variousmethods for the detection of proteins are well known in the art and areemployed for the detection of 108P5H8-related proteins and cells thatexpress 108P5H8-related proteins.

[0249] 108P5H8 expression analysis is also useful as a tool foridentifying and evaluating agents that modulate 108P5H8 gene expression.For example, 108P5H8 expression is significantly upregulated in prostatecancer, and is expressed in cancers of the tissues listed in Table I.Identification of a molecule or biological agent that inhibits 108P5H8expression or over-expression in cancer cells is of therapeutic value.For example, such an agent can be identified by using a screen thatquantifies 108P5H8 expression by RT-PCR, nucleic acid hybridization orantibody binding.

[0250] VIII.) Methods for Monitoring the Status of 108P5H8-Related Genesand Their Products

[0251] Oncogenesis is known to be a multistep process where cellulargrowth becomes progressively dysregulated and cells progress from anormal physiological state to precancerous and then cancerous states(see, e.g., Alers et al., Lab Invest. 77(5): 437-438 (1997) and Isaacset al., Cancer Surv. 23: 19-32 (1995)). In this context, examining abiological sample for evidence of dysregulated cell growth (such asaberrant 108P5H8 expression in cancers) allows for early detection ofsuch aberrant physiology, before a pathologic state such as cancer hasprogressed to a stage that therapeutic options are more limited and orthe prognosis is worse. In such examinations, the status of 108P5H8 in abiological sample of interest can be compared, for example, to thestatus of 108P5H8 in a corresponding normal sample (e.g. a sample fromthat individual or alternatively another individual that is not affectedby a pathology). An alteration in the status of 108P5H8 in thebiological sample (as compared to the normal sample) provides evidenceof dysregulated cellular growth. In addition to using a biologicalsample that is not affected by a pathology as a normal sample, one canalso use a predetermined normative value such as a predetermined normallevel of mRNA expression (see, e.g., Grever et al., J. Comp. Neurol.Dec. 9, 1996; 376(2): 306-14 and U.S. Pat. No. 5,837,501) to compare108P5H8 status in a sample.

[0252] The term “status” in this context is used according to its artaccepted meaning and refers to the condition or state of a gene and itsproducts. Typically, skilled artisans use a number of parameters toevaluate the condition or state of a gene and its products. Theseinclude, but are not limited to the location of expressed gene products(including the location of 108P5H8 expressing cells) as well as thelevel, and biological activity of expressed gene products (such as108P5H8 mRNA, polynucleotides and polypeptides). Typically, analteration in the status of 108P5H8 comprises a change in the locationof 108P5H8 and/or 108P5H8 expressing cells and/or an increase in 108P5H8mRNA and/or protein expression.

[0253] 108P5H8 status in a sample can be analyzed by a number of meanswell known in the art, including without limitation, immunohistochemicalanalysis, in situ hybridization, RT-PCR analysis on laser capturemicro-dissected samples, Western blot analysis, and tissue arrayanalysis. Typical protocols for evaluating the status of a 108P5H8 geneand gene products are found, for example in Ausubel et al. eds., 1995,Current Protocols In Molecular Biology, Units 2 (Northern Blotting), 4(Southern Blotting), 15 (Immunoblotting) and 18 (PCR Analysis). Thus,the status of 108P5H8 in a biological sample is evaluated by variousmethods utilized by skilled artisans including, but not limited togenomic Southern analysis (to examine, for example perturbations in a108P5H8 gene), Northern analysis and/or PCR analysis of 108P5H8 mRNA (toexamine, for example alterations in the polynucleotide sequences orexpression levels of 108P5H8 mRNAs), and, Western and/orimmunohistochemical analysis (to examine, for example alterations inpolypeptide sequences, alterations in polypeptide localization within asample, alterations in expression levels of 108P5H8 proteins and/orassociations of 108P5H8 proteins with polypeptide binding partners).Detectable 108P5H8 polynucleotides include, for example, a 108P5H8 geneor fragment thereof, 108P5H8 mRNA, alternative splice variants, 108P5H8mRNAs, and recombinant DNA or RNA molecules containing a 108P5H8polynucleotide.

[0254] The expression profile of 108P5H8 makes it a diagnostic markerfor local and/or metastasized disease, and provides information on thegrowth or oncogenic potential of a biological sample. In particular, thestatus of 108P5H8 provides information useful for predictingsusceptibility to particular disease stages, progression, and/or tumoraggressiveness. The invention provides methods and assays fordetermining 108P5H8 status and diagnosing cancers that express 108P5H8,such as cancers of the tissues listed in Table I. For example, because108P5H8 mRNA is so highly expressed in prostate and other cancersrelative to normal prostate tissue, assays that evaluate the levels of108P5H8 mRNA transcripts or proteins in a biological sample can be usedto diagnose a disease associated with 108P5H8 dysregulation, and canprovide prognostic information useful in defining appropriatetherapeutic options.

[0255] The expression status of 108P5H8 provides information includingthe presence, stage and location of dysplastic, precancerous andcancerous cells, predicting susceptibility to various stages of disease,and/or for gauging tumor aggressiveness. Moreover, the expressionprofile makes it useful as an imaging reagent for metastasized disease.Consequently, an aspect of the invention is directed to the variousmolecular prognostic and diagnostic methods for examining the status of108P5H8 in biological samples such as those from individuals sufferingfrom, or suspected of suffering from a pathology characterized bydysregulated cellular growth, such as cancer.

[0256] As described above, the status of 108P5H8 in a biological samplecan be examined by a number of well-known procedures in the art. Forexample, the status of 108P5H8 in a biological sample taken from aspecific location in the body can be examined by evaluating the samplefor the presence or absence of 108P5H8 expressing cells (e.g. those thatexpress 108P5H8 mRNAs or proteins): This examination can provideevidence of dysregulated cellular growth, for example, when108P5H8-expressing cells are found in a biological sample that does notnormally contain such cells (such as a lymph node), because suchalterations in the status of 108P5H8 in a biological sample are oftenassociated with dysregulated cellular growth. Specifically, oneindicator of dysregulated cellular growth is the metastases of cancercells from an organ of origin (such as the prostate) to a different areaof the body (such as a lymph node). In this context, evidence ofdysregulated cellular growth is important for example because occultlymph node metastases can be detected in a substantial proportion ofpatients with prostate cancer, and such metastases are associated withknown predictors of disease progression (see, e.g., Murphy et al.,Prostate 42(4): 315-317 (2000);Su et al., Semin. Surg. Oncol. 18(1):17-28 (2000) and Freeman et al., J Urol August 1995 154(2 Pt 1):474-8).

[0257] In one aspect, the invention provides methods for monitoring108P5H8 gene products by determining the status of 108P5H8 gene productsexpressed by cells from an individual suspected of having a diseaseassociated with dysregulated cell growth (such as hyperplasia or cancer)and then comparing the status so determined to the status of 108P5H8gene products in a corresponding normal sample. The presence of aberrant108P5H8 gene products in the test sample relative to the normal sampleprovides an indication of the presence of dysregulated cell growthwithin the cells of the individual.

[0258] In another aspect, the invention provides assays useful indetermining the presence of cancer in an individual, comprisingdetecting a significant increase in 108P5H8 mRNA or protein expressionin a test cell or tissue sample relative to expression levels in thecorresponding normal cell or tissue. The presence of 108P5H8 mRNA can,for example, be evaluated in tissue samples including but not limited tothose listed in Table I. The presence of significant 108P5H8 expressionin any of these tissues is useful to indicate the emergence, presenceand/or severity of a cancer, since the corresponding normal tissues donot express 108P5H8 mRNA or express it at lower levels.

[0259] In a related embodiment, 108P5H8 status is determined at theprotein level rather than at the nucleic acid level. For example, such amethod comprises determining the level of 108P5H8 protein expressed bycells in a test tissue sample and comparing the level so determined tothe level of 108P5H8 expressed in a corresponding normal sample. In oneembodiment, the presence of 108P5H8 protein is evaluated, for example,using immunohistochemical methods. 108P5H8 antibodies or bindingpartners capable of detecting 108P5H8 protein expression are used in avariety of assay formats well known in the art for this purpose.

[0260] In a further embodiment, one can evaluate the status of 108P5H8nucleotide and amino acid sequences in a biological sample in order toidentify perturbations in the structure of these molecules. Theseperturbations can include insertions, deletions, substitutions and thelike. Such evaluations are useful because perturbations in thenucleotide and amino acid sequences are observed in a large number ofproteins associated with a growth dysregulated phenotype (see, e.g.,Marrogi et al., 1999, J. Cutan. Pathol. 26(8):369-378). For example, amutation in the sequence of 108P5H8 may be indicative of the presence orpromotion of a tumor. Such assays therefore have diagnostic andpredictive value where a mutation in 108P5H8 indicates a potential lossof function or increase in tumor growth.

[0261] A wide variety of assays for observing perturbations innucleotide and amino acid sequences are well known in the art. Forexample, the size and structure of nucleic acid or amino acid sequencesof 108P5H8 gene products are observed by the Northern, Southern,Western, PCR and DNA sequencing protocols discussed herein. In addition,other methods for observing perturbations in nucleotide and amino acidsequences such as single strand conformation polymorphism analysis arewell known in the art (see, e.g., U.S. Pat. Nos. 5,382,510 issued Sep.7, 1999, and 5,952,170 issued Jan. 17, 1995).

[0262] Additionally, one can examine the methylation status of a 108P5H8gene in a biological sample. Aberrant demethylation and/orhypermethylation of CpG islands in gene 5′ regulatory regions frequentlyoccurs in immortalized and transformed cells, and can result in alteredexpression of various genes. For example, promoter hypermethylation ofthe pi-class glutathione S-transferase (a protein expressed in normalprostate but not expressed in >90% of prostate carcinomas) appears topermanently silence transcription of this gene and is the mostfrequently detected genomic alteration in prostate carcinomas (De Marzoet al., Am. J. Pathol. 155(6): 1985-1992 (1999)). In addition, thisalteration is present in at least 70% of cases of high-grade prostaticintraepithelial neoplasia (PIN) (Brooks et al., Cancer Epidemiol.Biomarkers Prev., 1998, 7:531-536). In another example, expression ofthe LAGE-I tumor specific gene (which is not expressed in normalprostate but is expressed in 25-50% of prostate cancers) is induced bydeoxy-azacytidine in lymphoblastoid cells, suggesting that tumoralexpression is due to demethylation (Lethe et al., Int. J. Cancer 76(6):903-908 (1998)). A variety of assays for examining methylation status ofa gene are well known in the art. For example, one can utilize, inSouthern hybridization approaches, methylation-sensitive restrictionenzymes that cannot cleave sequences that contain methylated CpG sitesto assess the methylation status of CpG islands. In addition, MSP(methylation specific PCR) can rapidly profile the methylation status ofall the CpG sites present in a CpG island of a given gene. Thisprocedure involves initial modification of DNA by sodium bisulfite(which will convert all unmethylated cytosines to uracil) followed byamplification using primers specific for methylated versus unmethylatedDNA. Protocols involving methylation interference can also be found forexample in Current Protocols In Molecular Biology, Unit 12, Frederick M.Ausubel et al. eds., 1995.

[0263] Gene amplification is an additional method for assessing thestatus of 108P5H8. Gene amplification is measured in a sample directly,for example, by conventional Southern blotting or Northern blotting toquantitate the transcription of mRNA (Thomas, 1980, Proc. Natl. Acad.Sci. USA, 77:5201-5205), dot blotting (DNA analysis), or in situhybridization, using an appropriately labeled probe, based on thesequences provided herein. Alternatively, antibodies are employed thatrecognize specific duplexes, including DNA duplexes, RNA duplexes, andDNA-RNA hybrid duplexes or DNA-protein duplexes. The antibodies in turnare labeled and the assay carried out where the duplex is bound to asurface, so that upon the formation of duplex on the surface, thepresence of antibody bound to the duplex can be detected.

[0264] Biopsied tissue or peripheral blood can be conveniently assayedfor the presence of cancer cells using for example, Northern, dot blotor RT-PCR analysis to detect 108P5H8 expression. The presence of RT-PCRamplifiable 108P5H8 mRNA provides an indication of the presence ofcancer. RT-PCR assays are well known in the art. RT-PCR detection assaysfor tumor cells in peripheral blood are currently being evaluated foruse in the diagnosis and management of a number of human solid tumors.In the prostate cancer field, these include RT-PCR assays for thedetection of cells expressing PSA and PSM (Verkaik et al., 1997, Urol.Res. 25:373-384; Ghossein et al., 1995, J. Clin. Oncol. 13:1195-2000;Heston et al., 1995, Clin. Chem. 41:1687-1688).

[0265] A further aspect of the invention is an assessment of thesusceptibility that an individual has for developing cancer. In oneembodiment, a method for predicting susceptibility to cancer comprisesdetecting 108P5H8 mRNA or 108P5H8 protein in a tissue sample, itspresence indicating susceptibility to cancer, wherein the degree of108P5H8 mRNA expression correlates to the degree of susceptibility. In aspecific embodiment, the presence of 108P5H8 in prostate or other tissueis examined, with the presence of 108P5H8 in the sample providing anindication of prostate cancer susceptibility (or the emergence orexistence of a prostate tumor). Similarly, one can evaluate theintegrity 108P5H8 nucleotide and amino acid sequences in a biologicalsample, in order to identify perturbations in the structure of thesemolecules such as insertions, deletions, substitutions and the like. Thepresence of one or more perturbations in 108P5H8 gene products in thesample is an indication of cancer susceptibility (or the emergence orexistence of a tumor).

[0266] The invention also comprises methods for gauging tumoraggressiveness. In one embodiment, a method for gauging aggressivenessof a tumor comprises determining the level of 108P5H8 mRNA or 108P5H8protein expressed by tumor cells, comparing the level so determined tothe level of 108P5H8 mRNA or 108P5H8 protein expressed in acorresponding normal tissue taken from the same individual or a normaltissue reference sample, wherein the degree of 108P5H8 mRNA or 108P5H8protein expression in the tumor sample relative to the normal sampleindicates the degree of aggressiveness. In a specific embodiment,aggressiveness of a tumor is evaluated by determining the extent towhich 108P5H8 is expressed in the tumor cells, with higher expressionlevels indicating more aggressive tumors. Another embodiment is theevaluation of the integrity of 108P5H8 nucleotide and amino acidsequences in a biological sample, in order to identify perturbations inthe structure of these molecules such as insertions, deletions,substitutions and the like. The presence of one or more perturbationsindicates more aggressive tumors.

[0267] Another embodiment of the invention is directed to methods forobserving the progression of a malignancy in an individual over time. Inone embodiment, methods for observing the progression of a malignancy inan individual over time comprise determining the level of 108P5H8 mRNAor 108P5H8 protein expressed by cells in a sample of the tumor,comparing the level so determined to the level of 108P5H8 mRNA or108P5H8 protein expressed in an equivalent tissue sample taken from thesame individual at a different time, wherein the degree of 108P5H8 mRNAor 108P5H8 protein expression in the tumor sample over time providesinformation on the progression of the cancer. In a specific embodiment,the progression of a cancer is evaluated by determining 108P5H8expression in the tumor cells over time, where increased expression overtime indicates a progression of the cancer. Also, one can evaluate theintegrity 108P5H8 nucleotide and amino acid sequences in a biologicalsample in order to identify perturbations in the structure of thesemolecules such as insertions, deletions, substitutions and the like,where the presence of one or more perturbations indicates a progressionof the cancer.

[0268] The above diagnostic approaches can be combined with any one of awide variety of prognostic and diagnostic protocols known in the art.For example, another embodiment of the invention is directed to methodsfor observing a coincidence between the expression of 108P5H8 gene and108P5H8 gene products (or perturbations in 108P5H8 gene and 108P5H8 geneproducts) and a factor that is associated with malignancy, as a meansfor diagnosing and prognosticating the status of a tissue sample. A widevariety of factors associated with malignancy can be utilized, such asthe expression of genes associated with malignancy (e.g. PSA, PSCA andPSM expression for prostate cancer etc.) as well as gross cytologicalobservations (see, e.g., Bocking et al., 1984, Anal. Quant. Cytol.6(2):74-88; Epstein, 1995, Hum. Pathol. 26(2):223-9; Thorson et al.,1998, Mod. Pathol. 11(6):543-51; Baisden et al., 1999, Am. J. Surg.Pathol. 23(8):918-24). Methods for observing a coincidence between theexpression of 108P5H8 gene and 108P5H8 gene products (or perturbationsin 108P5H8 gene and 108P5H8 gene products) and another factor that isassociated with malignancy are useful, for example, because the presenceof a set of specific factors that coincide with disease providesinformation crucial for diagnosing and prognosticating the status of atissue sample.

[0269] In one embodiment, methods for observing a coincidence betweenthe expression of 108P5H8 gene and 108P5H8 gene products (orperturbations in 108P5H8 gene and 108P5H8 gene products) and anotherfactor associated with malignancy entails detecting the overexpressionof 108P5H8 mRNA or protein in a tissue sample, detecting theoverexpression of PSA mRNA or protein in a tissue sample (or PSCA or PSMexpression), and observing a coincidence of 108P5H8 mRNA or protein andPSA mRNA or protein overexpression (or PSCA or PSM expression). In aspecific embodiment, the expression of 108P5H8 and PSA mRNA in prostatetissue is examined, where the coincidence of 108P5H8 and PSA mRNAoverexpression in the sample indicates the existence of prostate cancer,prostate cancer susceptibility or the emergence or status of a prostatetumor.

[0270] Methods for detecting and quantifying the expression of 108P5H8mRNA or protein are described herein, and standard nucleic acid andprotein detection and quantification technologies are well known in theart. Standard methods for the detection and quantification of 108P5H8mRNA include in situ hybridization using labeled 108P5H8 riboprobes,Northern blot and related techniques using 108P5H8 polynucleotideprobes, RT-PCR analysis using primers specific for 108P5H8, and otheramplification type detection methods, such as, for example, branchedDNA, SISBA, TMA and the like. In a specific embodiment,semi-quantitative RT-PCR is used to detect and quantify 108P5H8 mRNAexpression. Any number of primers capable of amplifying 108P5H8 can beused for this purpose, including but not limited to the various primersets specifically described herein. In a specific embodiment, polyclonalor monoclonal antibodies specifically reactive with the wild-type108P5H8 protein can be used in an immunohistochemical assay of biopsiedtissue.

[0271] IX.) Identification of Molecules that Interact with 108P5H8

[0272] The 108P5H8 protein and nucleic acid sequences disclosed hereinallow a skilled artisan to identify proteins, small molecules and otheragents that interact with 108P5H8, as well as pathways activated by108P5H8 via any one of a variety of art accepted protocols. For example,one can utilize one of the so-called interaction trap systems (alsoreferred to as the “two-hybrid assay”). In such systems, moleculesinteract and reconstitute a transcription factor which directsexpression of a reporter gene, whereupon the expression of the reportergene is assayed. Other systems identify protein-protein interactions invivo through reconstitution of a eukaryotic transcriptional activator,see, e.g., U.S. Pat. Nos. 5,955,280 issued Sep. 21, 1999, 5,925,523issued Jul. 20, 1999, 5,846,722 issued Dec. 8, 1998 and 6,004,746 issuedDec. 21, 1999. Algorithms are also available in the art for genome-basedpredictions of protein function (see, e.g., Marcotte, et al., Nature402: Nov. 4, 1999, 83-86).

[0273] Alternatively one can screen peptide libraries to identifymolecules that interact with 108P5H8 protein sequences. In such methods,peptides that bind to 108P5H8 are identified by screening libraries thatencode a random or controlled collection of amino acids. Peptidesencoded by the libraries are expressed as fusion proteins ofbacteriophage coat proteins, the bacteriophage particles are thenscreened against the 108P5H8 protein(s).

[0274] Accordingly, peptides having a wide variety of uses, such astherapeutic, prognostic or diagnostic reagents, are thus identifiedwithout any prior information on the structure of the expected ligand orreceptor molecule. Typical peptide libraries and screening methods thatcan be used to identify molecules that interact with 108P5H8 proteinsequences are disclosed for example in U.S. Pat. Nos. 5,723,286 issuedMar. 3, 1998 and 5,733,731 issued Mar. 31, 1998.

[0275] Alternatively, cell lines that express 108P5H8 are used toidentify protein-protein interactions mediated by 108P5H8. Suchinteractions can be examined using immunoprecipitation techniques (see,e.g., Hamilton B. J., et al. Biochem. Biophys. Res. Commun. 1999,261:646-51). 108P5H8 protein can be immunoprecipitated from108P5H8-expressing cell lines using anti-108P5H8 antibodies.Alternatively, antibodies against His-tag can be used in a cell lineengineered to express fusions of 108P5H8 and a His-tag (vectorsmentioned above). The immunoprecipitated complex can be examined forprotein association by procedures such as Western blotting,³⁵S-methionine labeling of proteins, protein microsequencing, silverstaining and two-dimensional gel electrophoresis.

[0276] Small molecules and ligands that interact with 108P5H8 can beidentified through related embodiments of such screening assays. Forexample, small molecules can be identified that interfere with proteinfunction, including molecules that interfere with 108P5H8's ability tomediate phosphorylation and de-phosphorylation, interaction with DNA orRNA molecules as an indication of regulation of cell cycles, secondmessenger signaling or tumorigenesis. Similarly, small molecules thatmodulate 108P5H8-related ion channel, protein pump, or cellcommunication functions are identified and used to treat patients thathave a cancer that expresses 108P5H8 (see, e.g., Hille, B., IonicChannels of Excitable Membranes 2^(nd) Ed., Sinauer Assoc., Sunderland,Mass., 1992). Moreover, ligands that regulate 108P5H8 function can beidentified based on their ability to bind 108P5H8 and activate areporter construct. Typical methods are discussed for example in U.S.Pat. No. 5,928,868 issued Jul. 27, 1999, and include methods for forminghybrid ligands in which at least one ligand is a small molecule. In anillustrative embodiment, cells engineered to express a fusion protein of108P5H8 and a DNA-binding protein are used to co-express a fusionprotein of a hybrid ligand/small molecule and a cDNA librarytranscriptional activator protein. The cells further contain a reportergene, the expression of which is conditioned on the proximity of thefirst and second fusion proteins to each other, an event that occursonly if the hybrid ligand binds to target sites on both hybrid proteins.Those cells that express the reporter gene are selected and the unknownsmall molecule or the unknown ligand is identified. This method providesa means of identifying modulators which activate or inhibit 108P5H8.

[0277] An embodiment of this invention comprises a method of screeningfor a molecule that interacts with an 108P5H8 amino acid sequence shownin FIG. 2 or FIG. 3, comprising the steps of contacting a population ofmolecules with a 108P5H8 amino acid sequence, allowing the population ofmolecules and the 108P5H8 amino acid sequence to interact underconditions that facilitate an interaction, determining the presence of amolecule that interacts with the 108P5H8 amino acid sequence, and thenseparating molecules that do not interact with the 108P5H8 amino acidsequence from molecules that do. In a specific embodiment, the methodfurther comprises purifying, characterizing and identifying a moleculethat interacts with the 108P5H8 amino acid sequence. The identifiedmolecule can be used to modulate a function performed by 108P5H8. In apreferred embodiment, the 108P5H8 amino acid sequence is contacted witha library of peptides.

[0278] X.) Therapeutic Methods and Compositions

[0279] The identification of 108P5H8 as a protein that is normallyexpressed in a restricted set of tissues, but which is also expressed inprostate and other cancers, opens a number of therapeutic approaches tothe treatment of such cancers. As contemplated herein, 108P5H8 functionsas a transcription factor involved in activating tumor-promoting genesor repressing genes that block tumorigenesis.

[0280] Accordingly, therapeutic approaches that inhibit the activity ofa 108P5H8 protein are useful for patients suffering from a cancer thatexpresses 108P5H8. These therapeutic approaches generally fall into twoclasses. One class comprises various methods for inhibiting the bindingor association of a 108P5H8 protein with its binding partner or withother proteins. Another class comprises a variety of methods forinhibiting the transcription of a 108P5H8 gene or translation of 108P5H8mRNA.

[0281] X.A.) Anti-Cancer Vaccines

[0282] The invention provides cancer vaccines comprising a108P5H8-related protein or 108P5H8-related nucleic acid. In view of theexpression of 108P5H8, cancer vaccines prevent and/or treat108P5H8-expressing cancers with minimal or no effects on non-targettissues. The use of a tumor antigen in a vaccine that generates humoraland/or cell-mediated immune responses as anti-cancer therapy is wellknown in the art and has been employed in prostate cancer using humanPSMA and rodent PAP immunogens (Hodge et al., 1995, Int. J. Cancer63:231-237; Fong et al., 1997, J. Immunol. 159:3113-3117).

[0283] Such methods can be readily practiced by employing a108P5H8-related protein, or an 108P5H8-encoding nucleic acid moleculeand recombinant vectors capable of expressing and presenting the 108P5H8immunogen (which typically comprises a number of antibody or T cellepitopes). Skilled artisans understand that a wide variety of vaccinesystems for delivery of immunoreactive epitopes are known in the art(see, e.g., Heryln et al., Ann Med February 1999 31(1):66-78; Maruyamaet al., Cancer Immunol Immunother June 2000 49(3):123-32) Briefly, suchmethods of generating an immune response (e.g. humoral and/orcell-mediated) in a mammal, comprise the steps of: exposing the mammal'simmune system to an immunoreactive epitope (e.g. an epitope present in a108P5H8 protein shown in FIG. 3 or analog or homolog thereof) so thatthe mammal generates an immune response that is specific for thatepitope (e.g. generates antibodies that specifically recognize thatepitope). In a preferred method, a 108P5H8 immunogen contains abiological motif, see e.g., Tables V-XVIII, XXII, and XXIII, or apeptide of a size range from 108P5H8 indicated in FIG. 5, FIG. 6, FIG.7, FIG. 8, and FIG. 9.

[0284] The entire 108P5H8 protein, immunogenic regions or epitopesthereof can be combined and delivered by various means. Such vaccinecompositions can include, for example, lipopeptides (e.g., Vitiello, A.et al., J. Clin. Invest. 95:341, 1995), peptide compositionsencapsulated in poly(DL-lactide-co-glycolide) (“PLG”) microspheres (see,e.g., Eldridge, et al., Molec. Immunol. 28:287-294, 1991: Alonso et al.,Vaccine 12:299-306, 1994; Jones et al., Vaccine 13:675-681, 1995),peptide compositions contained in immune stimulating complexes (ISCOMS)(see, e.g., Takahashi et al., Nature 344:873-875, 1990; Hu et al., ClinExp Immunol. 113:235-243, 1998), multiple antigen peptide systems (MAPs)(see e.g., Tam, J. P., Proc. Natl. Acad. Sci. U.S.A. 85:5409-5413, 1988;Tam, J. P., J. Immunol. Methods 196:17-32, 1996), peptides formulated asmultivalent peptides; peptides for use in ballistic delivery systems,typically crystallized peptides, viral delivery vectors (Perkus, M. E.et al., In: Concepts in vaccine development, Kaufmann, S. H. E., ed., p.379, 1996; Chakrabarti, S. et al., Nature 320:535, 1986; Hu, S. L. etal., Nature 320:537, 1986; Kieny, M. -P. et al., AIDS Bio/Technology4:790, 1986; Top, F. H. et al., J. Infect. Dis. 124:148, 1971; Chanda,P. K. et al., Virology 175:535, 1990), particles of viral or syntheticorigin (e.g., Kofler, N. et al., J. Immunol. Methods. 192:25, 1996;Eldridge, J. H. et al., Sem. Hematol. 30:16, 1993; Falo, L. D., Jr. etal., Nature Med. 7:649, 1995), adjuvants (Warren, H. S., Vogel, F. R.,and Chedid, L. A. Annu. Rev. Immunol. 4:369, 1986; Gupta, R. K. et al.,Vaccine 11:293, 1993), liposomes (Reddy, R. et al., J. Immunol.148:1585, 1992; Rock, K. L., Immunol. Today 17:131, 1996), or, naked orparticle absorbed cDNA (Ulmer, J. B. et al., Science 259:1745, 1993;Robinson, H. L., Hunt, L. A., and Webster, R. G., Vaccine 11:957, 1993;Shiver, J. W. et al., In: Concepts in vaccine development, Kaufmann, S.H. E., ed., p. 423, 1996; Cease, K. B., and Berzofsky, J. A., Annu. Rev.Immunol. 12:923, 1994 and Eldridge, J. H. et al., Sem. Hematol. 30:16,1993). Toxin-targeted delivery technologies, also known as receptormediated targeting, such as those of Avant Immunotherapeutics, Inc.(Needham, Mass.) may also be used.

[0285] In patients with 108P5H8-associated cancer, the vaccinecompositions of the invention can also be used in conjunction with othertreatments used for cancer, e.g., surgery, chemotherapy, drug therapies,radiation therapies, etc. including use in combination with immuneadjuvants such as IL-2, IL-12, GM-CSF, and the like.

[0286] Cellular Vaccines:

[0287] CTL epitopes can be determined using specific algorithms toidentify peptides within 108P5H8 protein that bind corresponding HLAalleles (see e.g., Table IV; Epimer™ and Epimatrix™, Brown University(URL www.brown.edu/Research/TB-HIV_Lab/epimatrix/epimatrix.html); and,BIMAS, (URL bimas.dcrt.nih.gov/; SYFPEITHI at URLsyfpeithi.bmi-heidelberg.com/). In a preferred embodiment, a 108P5H8immunogen contains one or more amino acid sequences identified usingtechniques well known in the art, such as the sequences shown in TablesV-XVIII, XXII, and XXIII or a peptide of 8, 9, 10 or 11 amino acidsspecified by an HLA Class I motif/supermotif (e.g., Table IV (A), TableIV (D), or Table IV (E)) and/or a peptide of at least 9 amino acids thatcomprises an HLA Class II motif/supermotif (e.g., Table IV (B) or TableIV (C)). As is appreciated in the art, the HLA Class I binding groove isessentially closed ended so that peptides of only a particular sizerange can fit into the groove and be bound, generally HLA Class Iepitopes are 8, 9, 10, or 11 amino acids long. In contrast, the HLAClass II binding groove is essentially open ended; therefore a peptideof about 9 or more amino acids can be bound by an HLA Class II molecule.Due to the binding groove differences between HLA Class I and II, HLAClass I motifs are length specific, i.e., position two of a Class Imotif is the second amino acid in an amino to carboxyl direction of thepeptide. The amino acid positions in a Class II motif are relative onlyto each other, not the overall peptide, i.e., additional amino acids canbe attached to the amino and/or carboxyl termini of a motif-bearingsequence. HLA Class II epitopes are often 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids long, or longer than25 amino acids.

[0288] Antibody-Based Vaccines

[0289] A wide variety of methods for generating an immune response in amammal are known in the art (for example as the first step in thegeneration of hybridomas). Methods of generating an immune response in amammal comprise exposing the mammal's immune system to an immunogenicepitope on a protein (e.g. a 108P5H8 protein) so that an immune responseis generated. A typical embodiment consists of a method for generatingan immune response to 108P5H8 in a host, by contacting the host with asufficient amount of at least one 108P5H8 B cell or cytotoxic T-cellepitope or analog thereof; and at least one periodic interval thereafterre-contacting the host with the 108P5H8 B cell or cytotoxic T-cellepitope or analog thereof. A specific embodiment consists of a method ofgenerating an immune response against a 108P5H8-related protein or aman-made multiepitopic peptide comprising: administering 108P5H8immunogen (e.g. a 108P5H8 protein or a peptide fragment thereof, an108P5H8 fusion protein or analog etc.) in a vaccine preparation to ahuman or another mammal. Typically, such vaccine preparations furthercontain a suitable adjuvant (see, e.g., U.S. Pat. No. 6,146,635) or auniversal helper epitope such as a PADRE™ peptide (Epimmune Inc., SanDiego, Calif.; see, e.g., Alexander et al., J. Immunol. 2000 164(3);164(3): 1625-1633; Alexander et al., Immunity 1994 1(9): 751-761 andAlexander et al., Immunol. Res. 1998 18(2): 79-92). An alternativemethod comprises generating an immune response in an individual againsta 108P5H8 immunogen by: administering in vivo to muscle or skin of theindividual's body a DNA molecule that comprises a DNA sequence thatencodes an 108P5H8 immunogen, the DNA sequence operatively linked toregulatory sequences which control the expression of the DNA sequence;wherein the DNA molecule is taken up by cells, the DNA sequence isexpressed in the cells and an immune response is generated against theimmunogen (see, e.g., U.S. Pat. No. 5,962,428). Optionally a geneticvaccine facilitator such as anionic lipids; saponins; lectins;estrogenic compounds; hydroxylated lower alkyls; dimethyl sulfoxide; andurea is also administered. In addition, an antiidiotypic antibody can beadministered that mimics 108P5H8, in order to generate a response to thetarget antigen.

[0290] Nucleic Acid Vaccines:

[0291] Vaccine compositions of the invention include nucleicacid-mediated modalities. DNA or RNA that encode protein(s) of theinvention can be administered to a patient. Genetic immunization methodscan be employed to generate prophylactic or therapeutic humoral andcellular immune responses directed against cancer cells expressing108P5H8. Constructs comprising DNA encoding a 108P5H8-relatedprotein/immunogen and appropriate regulatory sequences can be injecteddirectly into muscle or skin of an individual, such that the cells ofthe muscle or skin take-up the construct and express the encoded 108P5H8protein/immunogen. Alternatively, a vaccine comprises a 108P5H8-relatedprotein. Expression of the 108P5H8-related protein immunogen results inthe generation of prophylactic or therapeutic humoral and cellularimmunity against cells that bear a 108P5H8 protein. Various prophylacticand therapeutic genetic immunization techniques known in the art can beused (for review, see information and references published at Internetaddress www.genweb.com). Nucleic acid-based delivery is described, forinstance, in Wolff et. al., Science 247:1465 (1990) as well as U.S. Pat.Nos. 5,580,859; 5,589,466; 5,804,566; 5,739,118; 5,736,524; 5,679,647;WO 98/04720. Examples of DNA-based delivery technologies include “nakedDNA”, facilitated (bupivicaine, polymers, peptide-mediated) delivery,cationic lipid complexes, and particle-mediated (“gene gun”) orpressure-mediated delivery (see, e.g. U.S. Pat. No. 5,922,687).

[0292] For therapeutic or prophylactic immunization purposes, proteinsof the invention can be expressed via viral or bacterial vectors.Various viral gene delivery systems that can be used in the practice ofthe invention include, but are not limited to, vaccinia, fowlpox,canarypox, adenovirus, influenza, poliovirus, adeno-associated virus,lentivirus, and sindbis virus (see, e.g., Restifo, 1996, Curr. Opin.Immunol. 8:658-663; Tsang et al. J. Natl. Cancer Inst. 87:982-990(1995)). Non-viral delivery systems can also be employed by introducingnaked DNA encoding a 108P5H8-related protein into the patient (e.g.,intramuscularly or intradermally) to induce an anti-tumor response.

[0293] Vaccinia virus is used, for example, as a vector to expressnucleotide sequences that encode the peptides of the invention. Uponintroduction into a host, the recombinant vaccinia virus expresses theprotein immunogenic peptide, and thereby elicits a host immune response.Vaccinia vectors and methods useful in immunization protocols aredescribed in, e.g., U.S. Pat. No. 4,722,848. Another vector is BCG(Bacille Calmette Guerin). BCG vectors are described in Stover et al.,Nature 351:456-460 (1991). A wide variety of other vectors useful fortherapeutic administration or immunization of the peptides of theinvention, e.g. adeno and adeno-associated virus vectors, retroviralvectors, Salmonella typhi vectors, detoxified anthrax toxin vectors, andthe like, will be apparent to those skilled in the art from thedescription herein.

[0294] Thus, gene delivery systems are used to deliver a 108P5H8-relatednucleic acid molecule. In one embodiment, the full-length human 108P5H8cDNA is employed. In another embodiment, 108P5H8 nucleic acid moleculesencoding specific cytotoxic T lymphocyte (CTL) and/or antibody epitopesare employed.

[0295] Ex Vivo Vaccines

[0296] Various ex vivo strategies can also be employed to generate animmune response. One approach involves the use of antigen presentingcells (APCs) such as dendritic cells (DC) to present 108P5H8 antigen toa patient's immune system. Dendritic cells express MHC class I and IImolecules, B7 co-stimulator, and IL-12, and are thus highly specializedantigen presenting cells. In prostate cancer, autologous dendritic cellspulsed with peptides of the prostate-specific membrane antigen (PSMA)are being used in a Phase I clinical trial to stimulate prostate cancerpatients' immune systems (Tjoa et al., 1996, Prostate 28:65-69; Murphyet al., 1996, Prostate 29:371-380). Thus, dendritic cells can be used topresent 108P5H8 peptides to T cells in the context of MHC class I or IImolecules. In one embodiment, autologous dendritic cells are pulsed with108P5H8 peptides capable of binding to MHC class I and/or class IImolecules. In another embodiment, dendritic cells are pulsed with thecomplete 108P5H8 protein. Yet another embodiment involves engineeringthe overexpression of a 108P5H8 gene in dendritic cells using variousimplementing vectors known in the art, such as adenovirus (Arthur etal., 1997, Cancer Gene Ther. 4:17-25), retrovirus (Henderson et al.,1996, Cancer Res. 56:3763-3770), lentivirus, adeno-associated virus, DNAtransfection (Ribas et al., 1997, Cancer Res. 57:2865-2869), ortumor-derived RNA transfection (Ashley et al., 1997, J. Exp. Med.186:1177-1182). Cells that express 108P5H8 can also be engineered toexpress immune modulators, such as GM-CSF, and used as immunizingagents.

[0297] X.B.) 108P5H8 as a Target for Antibody-Based Therapy

[0298] 108P5H8 is an attractive target for antibody-based therapeuticstrategies. A number of antibody strategies are known in the art fortargeting both extracellular and intracellular molecules (see, e.g.,complement and ADCC mediated killing as well as the use of intrabodies).Because 108P5H8 is expressed by cancer cells of various lineagesrelative to corresponding normal cells, systemic administration of108P5H8-immunoreactive compositions are prepared that exhibit excellentsensitivity without toxic, non-specific and/or non-target effects causedby binding of the immunoreactive composition to non-target organs andtissues. Antibodies specifically reactive with domains of 108P5H8 areuseful to treat 108P5H8-expressing cancers systemically, either asconjugates with a toxin or therapeutic agent, or as naked antibodiescapable of inhibiting cell proliferation or function.

[0299] 108P5H8 antibodies can be introduced into a patient such that theantibody binds to 108P5H8 and modulates a function, such as aninteraction with a binding partner, and consequently mediatesdestruction of the tumor cells and/or inhibits the growth of the tumorcells. Mechanisms by which such antibodies exert a therapeutic effectcan include complement-mediated cytolysis, antibody-dependent cellularcytotoxicity, modulation of the physiological function of 108P5H8,inhibition of ligand binding or signal transduction pathways, modulationof tumor cell differentiation, alteration of tumor angiogenesis factorprofiles, and/or apoptosis.

[0300] Those skilled in the art understand that antibodies can be usedto specifically target and bind immunogenic molecules such as animmunogenic region of a 108P5H8 sequence shown in FIG. 2 or FIG. 3. Inaddition, skilled artisans understand that it is routine to conjugateantibodies to cytotoxic agents (see, e.g., Slevers et al. Blood 93:113678-3684 (Jun. 1, 1999)). When cytotoxic and/or therapeutic agents aredelivered directly to cells, such as by conjugating them to antibodiesspecific for a molecule expressed by that cell (e.g. 108P5H8), thecytotoxic agent will exert its known biological effect (i.e.cytotoxicity) on those cells.

[0301] A wide variety of compositions and methods for usingantibody-cytotoxic agent conjugates to kill cells are known in the art.In the context of cancers, typical methods entail administering to ananimal having a tumor a biologically effective amount of a conjugatecomprising a selected cytotoxic and/or therapeutic agent linked to atargeting agent (e.g. an anti-108P5H8 antibody) that binds to a marker(e.g. 108P5H8) expressed, accessible to binding or localized on the cellsurfaces. A typical embodiment is a method of delivering a cytotoxicand/or therapeutic agent to a cell expressing 108P5H8, comprisingconjugating the cytotoxic agent to an antibody that immunospecificallybinds to a 108P5H8 epitope, and, exposing the cell to the antibody-agentconjugate. Another illustrative embodiment is a method of treating anindividual suspected of suffering from metastasized cancer, comprising astep of administering parenterally to said individual a pharmaceuticalcomposition comprising a therapeutically effective amount of an antibodyconjugated to a cytotoxic and/or therapeutic agent.

[0302] Cancer immunotherapy using anti-108P5H8 antibodies can be done inaccordance with various approaches that have been successfully employedin the treatment of other types of cancer, including but not limited tocolon cancer (Arlen et al., 1998, Crit. Rev. Immunol. 18:133-138),multiple myeloma (Ozaki et al., 1997, Blood 90:3179-3186, Tsunenari etal., 1997, Blood 90:2437-2444), gastric cancer (Kasprzyk et al., 1992,Cancer Res. 52:2771-2776), B-cell lymphoma (Funakoshi et al., 1996, J.Immunother. Emphasis Tumor Immunol. 19:93-101), leukemia (Zhong et al.,1996, Leuk. Res. 20:581-589), colorectal cancer (Moun et al., 1994,Cancer Res. 54:6160-6166; Velders et al., 1995, Cancer Res.55:4398-4403), and breast cancer (Shepard et al., 1991, J. Clin.Immunol. 11:117-127). Some therapeutic approaches involve conjugation ofnaked antibody to a toxin or radioisotope, such as the conjugation ofY⁹¹ or I¹³¹ to anti-CD20 antibodies (e.g., Zevalin™, IDECPharmaceuticals Corp. or Bexxar™, Coulter Pharmaceuticals), while othersinvolve co-administration of antibodies and other therapeutic agents,such as Herceptin™ (trastuzumab) with paclitaxel (Genentech, Inc.). Theantibodies can be conjugated to a therapeutic agent. To treat prostatecancer, for example, 108P5H8 antibodies can be administered inconjunction with radiation, chemotherapy or hormone ablation. Also,antibodies can be conjugated to a toxin such as calicheamicin (e.g.,Mylotarg™, Wyeth-Ayerst, Madison, N.J., a recombinant humanized IgG₄kappa antibody conjugated to antitumor antibiotic calicheamicin) or amaytansinoid (e.g., taxane-based Tumor-Activated Prodrug, TAP, platform,ImmunoGen, Cambridge, Mass., also see e.g., U.S. Pat. No. 5,416,064).

[0303] Although 108P5H8 antibody therapy is useful for all stages ofcancer, antibody therapy can be particularly appropriate in advanced ormetastatic cancers. Treatment with the antibody therapy of the inventionis indicated for patients who have received one or more rounds ofchemotherapy. Alternatively, antibody therapy of the invention iscombined with a chemotherapeutic or radiation regimen for patients whohave not received chemotherapeutic treatment. Additionally, antibodytherapy can enable the use of reduced dosages of concomitantchemotherapy, particularly for patients who do not tolerate the toxicityof the chemotherapeutic agent very well. Fan et al. (Cancer Res.53:4637-4642, 1993), Prewett et al. (International J. of Onco.9:217-224, 1996), and Hancock et al. (Cancer Res. 51:4575-4580, 1991)describe the use of various antibodies together with chemotherapeuticagents.

[0304] Although 108P5H8 antibody therapy is useful for all stages ofcancer, antibody therapy can be particularly appropriate in advanced ormetastatic cancers. Treatment with the antibody therapy of the inventionis indicated for patients who have received one or more rounds ofchemotherapy. Alternatively, antibody therapy of the invention iscombined with a chemotherapeutic or radiation regimen for patients whohave not received chemotherapeutic treatment. Additionally, antibodytherapy can enable the use of reduced dosages of concomitantchemotherapy, particularly for patients who do not tolerate the toxicityof the chemotherapeutic agent very well.

[0305] Cancer patients can be evaluated for the presence and level of108P5H8 expression, preferably using immunohistochemical assessments oftumor tissue, quantitative 108P5H8 imaging, or other techniques thatreliably indicate the presence and degree of 108P5H8 expression.Immunohistochemical analysis of tumor biopsies or surgical specimens ispreferred for this purpose. Methods for immunohistochemical analysis oftumor tissues are well known in the art.

[0306] Anti-108P5H8 monoclonal antibodies that treat prostate and othercancers include those that initiate a potent immune response against thetumor or those that are directly cytotoxic. In this regard, anti-108P5H8monoclonal antibodies (mAbs) can elicit tumor cell lysis by eithercomplement-mediated or antibody-dependent cell cytotoxicity (ADCC)mechanisms, both of which require an intact Fc portion of theimmunoglobulin molecule for interaction with effector cell Fc receptorsites on complement proteins. In addition, anti-108P5H8 mAbs that exerta direct biological effect on tumor growth are useful to treat cancersthat express 108P5H8. Mechanisms by which directly cytotoxic mAbs actinclude: inhibition of cell growth, modulation of cellulardifferentiation, modulation of tumor angiogenesis factor profiles, andthe induction of apoptosis. The mechanism(s) by which a particularanti-108P5H8 mAb exerts an anti-tumor effect is evaluated using anynumber of in vitro assays that evaluate cell death such as ADCC, ADMMC,complement-mediated cell lysis, and so forth, as is generally known inthe art.

[0307] In some patients, the use of murine or other non-human monoclonalantibodies, or human/mouse chimeric mAbs can induce moderate to strongimmune responses against the non-human antibody. This can result inclearance of the antibody from circulation and reduced efficacy. In themost severe cases, such an immune response can lead to the extensiveformation of immune complexes which, potentially, can cause renalfailure. Accordingly, preferred monoclonal antibodies used in thetherapeutic methods of the invention are those that are either fullyhuman or humanized and that bind specifically to the target 108P5H8antigen with high affinity but exhibit low or no antigenicity in thepatient.

[0308] Therapeutic methods of the invention contemplate theadministration of single anti-108P5H8 mAbs as well as combinations, orcocktails, of different mAbs. Such mAb cocktails can have certainadvantages inasmuch as they contain mAbs that target different epitopes,exploit different effector mechanisms or combine directly cytotoxic mAbswith mAbs that rely on immune effector functionality. Such mAbs incombination can exhibit synergistic therapeutic effects. In addition,anti-108P5H8 mAbs can be administered concomitantly with othertherapeutic modalities, including but not limited to variouschemotherapeutic agents, androgen-blockers, immune modulators (e.g.,IL-2, GM-CSF), surgery or radiation. The anti-108P5H8 mAbs areadministered in their “naked” or unconjugated form, or can have atherapeutic agent(s) conjugated to them.

[0309] Anti-108P5H8 antibody formulations are administered via any routecapable of delivering the antibodies to a tumor cell. Routes ofadministration include, but are not limited to, intravenous,intraperitoneal, intramuscular, intratumor, intradermal, and the like.Treatment generally involves repeated administration of the anti-108P5H8antibody preparation, via an acceptable route of administration such asintravenous injection (IV), typically at a dose in the range of about0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9., 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 15, 20, or 25 mg/kg body weight. In general, doses in the range of10-1000 mg mAb per week are effective and well tolerated.

[0310] Based on clinical experience with the Herceptin™ mAb in thetreatment of metastatic breast cancer, an initial loading dose ofapproximately 4 mg/kg patient body weight IV, followed by weekly dosesof about 2 mg/kg IV of the anti-108P5H8 mAb preparation represents anacceptable dosing regimen. Preferably, the initial loading dose isadministered as a 90 minute or longer infusion. The periodic maintenancedose is administered as a 30 minute or longer infusion, provided theinitial dose was well tolerated. As appreciated by those of skill in theart, various factors can influence the ideal dose regimen in aparticular case. Such factors include, for example, the binding affinityand half life of the Ab or mAbs used, the degree of 108P5H8 expressionin the patient, the extent of circulating shed 108P5H8 antigen, thedesired steady-state antibody concentration level, frequency oftreatment, and the influence of chemotherapeutic or other agents used incombination with the treatment method of the invention, as well as thehealth status of a particular patient.

[0311] Optionally, patients should be evaluated for the levels of108P5H8 in a given sample (e.g. the levels of circulating 108P5H8antigen and/or 108P5H8 expressing cells) in order to assist in thedetermination of the most effective dosing regimen, etc. Suchevaluations are also used for monitoring purposes throughout therapy,and are useful to gauge therapeutic success in combination with theevaluation of other parameters (for example, urine cytology and/orImmunoCyt levels in bladder cancer therapy, or by analogy, serum PSAlevels in prostate cancer therapy).

[0312] Anti-idiotypic anti-108P5H8 antibodies can also be used inanti-cancer therapy as a vaccine for inducing an immune response tocells expressing a 108P5H8-related protein. In particular, thegeneration of anti-idiotypic antibodies is well known in the art; thismethodology can readily be adapted to generate anti-idiotypicanti-108P5H8 antibodies that mimic an epitope on a 108P5H8-relatedprotein (see, for example, Wagner et al., 1997, Hybridoma 16: 33-40;Foon et al., 1995, J. Clin. Invest. 96:334-342; Herlyn et al., 1996,Cancer Immunol. Immunother. 43:65-76). Such an anti-idiotypic antibodycan be used in cancer vaccine strategies.

[0313] X.C.) 108P5H8 as a Target for Cellular Immune Responses

[0314] Vaccines and methods of preparing vaccines that contain animmunogenically effective amount of one or more HLA-binding peptides asdescribed herein are further embodiments of the invention. Furthermore,vaccines in accordance with the invention encompass compositions of oneor more of the claimed peptides. A peptide can be present in a vaccineindividually. Alternatively, the peptide can exist as a homopolymercomprising multiple copies of the same peptide, or as a heteropolymer ofvarious peptides. Polymers have the advantage of increased immunologicalreaction and, where different peptide epitopes are used to make up thepolymer, the additional ability to induce antibodies and/or CTLs thatreact with different antigenic determinants of the pathogenic organismor tumor-related peptide targeted for an immune response. Thecomposition can be a naturally occurring region of an antigen or can beprepared, e.g., recombinantly or by chemical synthesis.

[0315] Carriers that can be used with vaccines of the invention are wellknown in the art, and include, e.g., thyroglobulin, albumins such ashuman serum albumin, tetanus toxoid, polyamino acids such as polyL-lysine, poly L-glutamic acid, influenza, hepatitis B virus coreprotein, and the like. The vaccines can contain a physiologicallytolerable (i.e., acceptable) diluent such as water, or saline,preferably phosphate buffered saline. The vaccines also typicallyinclude an adjuvant. Adjuvants such as incomplete Freund's adjuvant,aluminum phosphate, aluminum hydroxide, or alum are examples ofmaterials well known in the art. Additionally, as disclosed herein, CTLresponses can be primed by conjugating peptides of the invention tolipids, such as tripalmitoyl-S-glycerylcysteinlyseryl-serine (P₃CSS).Moreover, an adjuvant such as a syntheticcytosine-phosphorothiolated-guanine-containing (CpG) oligonucleotideshas been found to increase CTL responses 10- to 100-fold. (see, e.g.Davila and Celis J. Immunol. 165:539-547 (2000))

[0316] Upon immunization with a peptide composition in accordance withthe invention, via injection, aerosol, oral, transdermal, transmucosal,intrapleural, intrathecal, or other suitable routes, the immune systemof the host responds to the vaccine by producing large amounts of CTLsand/or HTLs specific for the desired antigen. Consequently, the hostbecomes at least partially immune to later development of cells thatexpress or overexpress 108P5H8 antigen, or derives at least sometherapeutic benefit when the antigen was tumor-associated.

[0317] In some embodiments, it may be desirable to combine the class Ipeptide components with components that induce or facilitateneutralizing antibody and or helper T cell responses directed to thetarget antigen. A preferred embodiment of such a composition comprisesclass I and class II epitopes in accordance with the invention. Analternative embodiment of such a composition comprises a class I and/orclass II epitope in accordance with the invention, along with a crossreactive HTL epitope such as PADRE™ (Epimmune, San Diego, Calif.)molecule (described e.g., in U.S. Pat. No. 5,736,142).

[0318] A vaccine of the invention can also include antigen-presentingcells (APC), such as dendritic cells (DC), as a vehicle topresent-peptides of the invention. Vaccine compositions can be createdin vitro, following dendritic cell mobilization and harvesting, wherebyloading of dendritic cells occurs in vitro. For example, dendritic cellsare transfected, e.g., with a minigene in accordance with the invention,or are pulsed with peptides. The dendritic cell can then be administeredto a patient to elicit immune responses in vivo. Vaccine compositions,either DNA- or peptide-based, can also be administered in vivo incombination with dendritic cell mobilization whereby loading ofdendritic cells occurs in vivo.

[0319] Preferably, the following principles are utilized when selectingan array of epitopes for inclusion in a polyepitopic composition for usein a vaccine, or for selecting discrete epitopes to be included in avaccine and/or to be encoded by nucleic acids such as a minigene. It ispreferred that each of the following principles be balanced in order tomake the selection. The multiple epitopes to be incorporated in a givenvaccine composition may be, but need not be, contiguous in sequence inthe native antigen from which the epitopes are derived.

[0320] 1.) Epitopes are selected which, upon administration, mimicimmune responses that have been observed to be correlated with tumorclearance. For HLA Class I this includes 3-4 epitopes that come from atleast one tumor associated antigen (TAA). For HLA Class II a similarrationale is employed; again 3-4 epitopes are selected from at least oneTAA (see, e.g., Rosenberg et al., Science 278:1447-1450). Epitopes fromone TAA may be used in combination with epitopes from one or moreadditional TAAs to produce a vaccine that targets tumors with varyingexpression patterns of frequently-expressed TAAs.

[0321] 2.) Epitopes are selected that have the requisite bindingaffinity established to be correlated with immunogenicity: for HLA ClassI an IC₅₀ of 500 nM or less, often 200 nM or less; and for Class II anIC₅₀ of 1000 nM or less.

[0322] 3.) Sufficient supermotif bearing-peptides, or a sufficient arrayof allele-specific motif-bearing peptides, are selected to give broadpopulation coverage. For example, it is preferable to have at least 80%population coverage. A Monte Carlo analysis, a statistical evaluationknown in the art, can be employed to assess the breadth, or redundancyof, population coverage.

[0323] 4.) When selecting epitopes from cancer-related antigens it isoften useful to select analogs because the patient may have developedtolerance to the native epitope.

[0324] 5.) Of particular relevance are epitopes referred to as “nestedepitopes.” Nested epitopes occur where at least two epitopes overlap ina given peptide sequence. A nested peptide sequence can comprise B cell,HLA class I and/or HLA class II epitopes. When providing nestedepitopes, a general objective is to provide the greatest number ofepitopes per sequence. Thus, an aspect is to avoid providing a peptidethat is any longer than the amino terminus of the amino terminal epitopeand the carboxyl terminus of the carboxyl terminal epitope in thepeptide. When providing a multi-epitopic sequence, such as a sequencecomprising nested epitopes, it is generally important to screen thesequence in order to insure that it does not have pathological or otherdeleterious biological properties.

[0325] 6.) If a polyepitopic protein is created, or when creating aminigene, an objective is to generate the smallest peptide thatencompasses the epitopes of interest. This principle is similar, if notthe same as that employed when selecting a peptide comprising nestedepitopes. However, with an artificial polyepitopic peptide, the sizeminimization objective is balanced against the need to integrate anyspacer sequences between epitopes in the polyepitopic protein. Spaceramino acid residues can, for example, be introduced to avoid junctionalepitopes (an epitope recognized by the immune system, not present in thetarget antigen and only created by the man-made juxtaposition ofepitopes), or to facilitate cleavage between epitopes and therebyenhance epitope presentation. Junctional epitopes are generally to beavoided because the recipient may generate an immune response to thatnon-native epitope. Of particular concern is a junctional epitope thatis a “dominant epitope.” A dominant epitope may lead to such a zealousresponse that immune responses to other epitopes are diminished orsuppressed.

[0326] 7.) Where the sequences of multiple variants of the same targetprotein are present, potential peptide epitopes can also be selected onthe basis of their conservancy. For example, a criterion for conservancymay define that the entire sequence of an HLA class I binding peptide orthe entire 9 -mer core of a class II binding peptide be conserved in adesignated percentage of the sequences-evaluated for a specific proteinantigen.

[0327] X.C.1. . Minigene Vaccines

[0328] A number of different approaches are available which allowsimultaneous delivery of multiple epitopes. Nucleic acids encoding thepeptides of the invention are a particularly useful embodiment of theinvention. Epitopes for inclusion in a minigene are preferably selectedaccording to the guidelines set forth in the previous section. Apreferred means of administering nucleic acids encoding the peptides ofthe invention uses minigene constructs encoding a peptide comprising oneor multiple epitopes of the invention.

[0329] The use of multi-epitope minigenes is described below and in,Ishioka et al., J. Immunol. 162:3915-3925, 1999; An, L. and Whitton, J.L., J. Virol. 71:2292, 1997; Thomson, S. A. et al., J. Immunol. 157:822,1996; Whitton, J. L. et al., J. Virol. 67:348, 1993; Hanke, R. et al.,Vaccine 16:426, 1998. For example, a multi-epitope DNA plasmid encodingsupermotif- and/or motif-beating epitopes derived 108P5H8, the PADRE®universal helper T cell epitope (or multiple HTL epitopes from 108P5H8)and an endoplasmic reticulum-translocating signal sequence can beengineered. A vaccine may also comprise epitopes that are derived fromother TAAs.

[0330] The immunogenicity of a multi-epitopic minigene can be confirmedin transgenic mice to evaluate the magnitude of CTL induction responsesagainst the epitopes tested. Further, the immunogenicity of DNA-encodedepitopes in vivo can be correlated with the in vitro responses ofspecific CTL lines against target cells transfected with the DNAplasmid. Thus, these experiments can show that the minigene serves toboth: 1.) generate a CTL response and 2.) that the induced CTLsrecognized cells expressing the encoded epitopes.

[0331] For example, to create a DNA sequence encoding the selectedepitopes (minigene) for expression in human cells, the amino acidsequences of the epitopes may be reverse translated. A human codon usagetable can be used to guide the codon choice for each amino acid. Theseepitope-encoding DNA sequences may be directly adjoined, so that whentranslated, a continuous polypeptide sequence is created. To optimizeexpression and/or immunogenicity, additional elements can beincorporated into the minigene design. Examples of amino acid sequencesthat can be reverse translated and included in the minigene sequenceinclude: HLA class I epitopes, HLA class II epitopes, antibody epitopes,a ubiquitination signal sequence, and/or an endoplasmic reticulumtargeting signal. In addition, HLA presentation of CTL and HTL epitopesmay be improved by including synthetic (e.g. poly-alanine) ornaturally-occurring flanking sequences adjacent to the CTL or HTLepitopes; these larger peptides comprising the epitope(s) are within thescope of the invention.

[0332] The minigene sequence may be converted to DNA by assemblingoligonucleotides that encode the plus and minus strands of the minigene.Overlapping oligonucleotides (30-100 bases long) may be synthesized,phosphorylated, purified and annealed under appropriate conditions usingwell known techniques. The ends of the oligonucleotides can be joined,for example, using T4 DNA ligase. This synthetic minigene, encoding theepitope polypeptide, can then be cloned into a desired expressionvector.

[0333] Standard regulatory sequences well known to those of skill in theart are preferably included in the vector to ensure expression in thetarget cells. Several vector elements are desirable: a promoter with adown-stream cloning site for minigene insertion; a polyadenylationsignal for efficient transcription termination; an E. coli origin ofreplication; and an E. coli selectable marker (e.g. ampicillin orkanamycin resistance). Numerous promoters can be used for this purpose,e.g., the human cytomegalovirus (hCMV) promoter. See, e.g., U.S. Pat.Nos. 5,580,859 and 5,589,466 for other suitable promoter sequences.

[0334] Additional vector modifications may be desired to optimizeminigene expression and immunogenicity. In some cases, introns arerequired for efficient gene expression, and one or more synthetic ornaturally-occurring introns could be incorporated into the transcribedregion of the minigene. The inclusion of mRNA stabilization sequencesand sequences for replication in mammalian cells may also be consideredfor increasing minigene expression.

[0335] Once an expression vector is selected, the minigene is clonedinto the polylinker region downstream of the promoter. This plasmid istransformed into an appropriate E. coli strain, and DNA is preparedusing standard techniques. The orientation and DNA sequence of theminigene, as well as all other elements included in the vector, areconfirmed using restriction mapping and DNA sequence analysis. Bacterialcells harboring the correct plasmid can be stored as a master cell bankand a working cell bank.

[0336] In addition, immunostimulatory sequences (ISSs or CpGs) appear toplay a role in the immunogenicity of DNA vaccines. These sequences maybe included in the vector, outside the minigene coding sequence, ifdesired to enhance immunogenicity.

[0337] In some embodiments, a bi-cistronic expression vector whichallows production of both the minigene-encoded epitopes and a secondprotein (included to enhance or decrease immunogenicity) can be used.Examples of proteins or polypeptides that could beneficially enhance theimmune response if co-expressed include cytokines (e.g., IL-2, IL-12,GM-CSF), cytokine-inducing molecules (e.g., LeIF), costimulatorymolecules, or for HTL responses, pan-DR binding proteins (PADRE™,Epimmune, San Diego, Calif.). Helper (HTL) epitopes can be joined tointracellular targeting signals and expressed separately from expressedCTL epitopes; this allows direction of the HTL epitopes to a cellcompartment different than that of the CTL epitopes. If required, thiscould facilitate more efficient entry of HTL epitopes into the HLA classII pathway, thereby improving HTL induction. In contrast to HTL or CTLinduction, specifically decreasing the immune response by co-expressionof immunosuppressive molecules (e.g. TGF-β) may be beneficial in certaindiseases.

[0338] Therapeutic quantities of plasmid DNA can be produced forexample, by fermentation in E. coli, followed by purification. Aliquotsfrom the working cell bank are used to inoculate growth medium, andgrown to saturation in shaker flasks or a bioreactor according towell-known techniques. Plasmid DNA can be purified using standardbioseparation technologies such as solid phase anion-exchange resinssupplied by QIAGEN, Inc. (Valencia, Calif.). If required, supercoiledDNA can be isolated from the open circular and linear forms using gelelectrophoresis or other methods.

[0339] Purified plasmid DNA can be prepared for injection using avariety of formulations. The simplest of these is reconstitution oflyophilized DNA in sterile phosphate-buffer saline (PBS). This approach,known as “naked DNA,” is currently being used for intramuscular (IM)administration in clinical trials. To maximize the immunotherapeuticeffects of minigene DNA vaccines, an alternative method for formulatingpurified plasmid DNA may be desirable. A variety of methods have beendescribed, and new techniques may become available. Cationic lipids,glycolipids, and fusogenic liposomes can also be used in the formulation(see, e.g:, as described by WO 93/24640; Mannino & Gould-Fogerite,BioTechniques 6(7): 682 (1988); U.S. Pat No. 5,279,833; WO 91/06309; andFelgner, et al., Proc. Nat'l Acad. Sci. USA 84:7413 (1987). In addition,peptides and compounds referred to collectively as protective,interactive, non-condensing compounds (PINC) could also be complexed topurified plasmid DNA to influence variables such as stability,intramuscular dispersion, or trafficking to specific organs or celltypes.

[0340] Target cell sensitization can be used as a functional assay forexpression and HLA class I presentation of minigene-encoded CTLepitopes. For example, the plasmid DNA is introduced into a mammaliancell line that is suitable as a target for standard CTL chromium releaseassays. The transfection method used will be dependent on the finalformulation. Electroporation can be used for “naked” DNA, whereascationic lipids allow direct in vitro transfection. A plasmid expressinggreen fluorescent protein (GFP) can be co-transfected to allowenrichment of transfected cells using fluorescence activated cellsorting (FACS). These cells are then chromium-51 (⁵¹Cr) labeled and usedas target cells for epitope-specific CTL lines; cytolysis, detected by⁵¹Cr release, indicates both production of, and HLA presentation of,minigene-encoded CTL epitopes. Expression of HTL epitopes may beevaluated in an analogous manner using assays to assess HTL activity.

[0341] In vivo immunogenicity is a second approach for functionaltesting of minigene DNA formulations. Transgenic mice expressingappropriate human HLA proteins are immunized with the DNA product. Thedose and route of administration are formulation dependent (e.g., IM forDNA in PBS, intraperitoneal (i.p.) for lipid-complexed DNA). Twenty-onedays after immunization, splenocytes are harvested and restimulated forone week in the presence of peptides encoding each epitope being tested.Thereafter, for CTL effector cells, assays are conducted for cytolysisof peptide-loaded, ⁵¹Cr-labeled target cells using standard techniques.Lysis of target cells that were sensitized by HLA loaded with peptideepitopes, corresponding to minigene-encoded epitopes, demonstrates DNAvaccine function for in vivo induction of CTLs. Immunogenicity of HTLepitopes is confirmed in transgenic mice in an analogous manner.

[0342] Alternatively, the nucleic acids can be administered usingballistic delivery as described, for instance, in U.S. Pat. No.5,204,253. Using this technique, particles comprised solely of DNA areadministered. In a further alternative embodiment, DNA can be adhered toparticles, such as gold particles.

[0343] Minigenes can also be delivered using other bacterial or viraldelivery systems well known in the art, e.g., an expression constructencoding epitopes of the invention can be incorporated into a viralvector such as vaccinia.

[0344] X.C.2. Combinations of CTL Peptides with Helper Peptides

[0345] Vaccine compositions comprising CTL peptides of the invention canbe modified, e.g., analoged, to provide desired attributes, such asimproved serum half life, broadened population coverage or enhancedimmunogenicity.

[0346] For instance, the ability of a peptide to induce CTL activity canbe enhanced by linking the peptide to a sequence which contains at leastone epitope that is capable of inducing a T helper cell response.Although a CTL peptide can be directly-linked to a T helper peptide,often CTL epitope/HTL epitope conjugates are linked by a spacermolecule. The spacer is typically comprised of relatively small, neutralmolecules, such as amino acids or amino acid mimetics, which aresubstantially uncharged under physiological conditions. The spacers aretypically selected from, e.g., Ala, Gly, or other neutral spacers ofnonpolar amino acids or neutral polar amino acids. It will be understoodthat the optionally present spacer need not be comprised of the sameresidues and thus may be a hetero- or homo-oligomer. When present, thespacer will usually be at least one or two residues, more usually threeto six residues and sometimes 10 or more residues. The CTL peptideepitope can be linked to the T helper peptide epitope either directly orvia a spacer either at the amino or carboxy terminus of the CTL peptide.The amino terminus of either the immunogenic peptide or the T helperpeptide may be acylated.

[0347] In certain embodiments, the T helper peptide is one that isrecognized by T helper cells present in a majority of a geneticallydiverse population. This can be accomplished by selecting peptides thatbind to many, most, or all of the HLA class II molecules. Examples ofsuch amino acid bind many HLA Class II molecules include sequences fromantigens such as tetanus toxoid at positions 830-843 (QYIKANSKFIGITE;SEQ ID NO: 2582), Plasmodium falciparum circumsporozoite (CS) protein atpositions 378-398 (DIEKKIAKMEKASSVFNVVNS; SEQ ID NO: 2583), andStreptococcus 18 kD protein at positions 116-131 (GAVDSILGGVATYGAA; SEQID NO: 2584). Other examples include peptides bearing a DR 1-4-7supermotif, or either of the DR3 motifs.

[0348] Alternatively, it is possible to prepare synthetic peptidescapable of stimulating T helper lymphocytes, in a loosely HLA-restrictedfashion, using amino acid sequences not found in nature (see, e.g., PCTpublication WO 95/07707). These synthetic compounds calledPan-DR-binding epitopes (e.g., PADRE™, Epimmune, Inc., San Diego,Calif.) are designed to most preferably bind most HLA-DR (human HLAclass II) molecules. For instance, a pan-DR-binding epitope peptidehaving the formula: aKXVAAWTLKAAa (SEQ ID NO: 2585), where “X” is eithercyclohexylalanine, phenylalanine, or tyrosine, and a is either D-alanineor L-alanine, has been found to bind to most HLA-DR alleles, and tostimulate the response of T helper lymphocytes from most individuals,regardless of their HLA type. An alternative of a pan-DR binding epitopecomprises all “L” natural amino acids and can be provided in the form ofnucleic acids that encode the epitope.

[0349] HTL peptide epitopes can also be modified to alter theirbiological properties. For example, they can be modified to includeD-amino acids to increase their resistance to proteases and thus extendtheir serum half life, or they can be conjugated to other molecules suchas lipids, proteins, carbohydrates, and the like to increase theirbiological activity. For example, a T helper peptide can be conjugatedto one or more palmitic acid chains at either the amino or carboxyltermini.

[0350] X.C.3. Combinations of CTL Peptides with T Cell Priming Agents

[0351] In some embodiments it may be desirable to include in thepharmaceutical compositions of the invention at least one componentwhich primes B lymphocytes or T lymphocytes. Lipids have been identifiedas agents capable of priming CTL in vivo. For example, palmitic acidresidues can be attached to the ε-and α-amino groups of a lysine residueand then linked, e.g., via one or more linking residues such as Gly,Gly-Gly-, Ser, Ser-Ser, or the like, to an immunogenic peptide. Thelipidated peptide can then be administered either directly in a micelleor particle, incorporated into a liposome, or emulsified in an adjuvant,e.g., incomplete Freund's adjuvant. In a preferred embodiment, aparticularly effective immunogenic composition comprises palmitic acidattached to ε- and α-amino groups of Lys, which is attached via linkage,e.g., Ser-Ser, to the amino terminus of the immunogenic peptide.

[0352] As another example of lipid priming of CTL responses, E. colilipoproteins, such as tripalmitoyl-S-glycerylcysteinlyseryl-serine(P₃CSS) can be used to prime virus specific CTL when covalently attachedto an appropriate peptide (see, e.g., Deres, et al., Nature 342:561,1989). Peptides of the invention can be coupled to P₃CSS, for example,and the lipopeptide administered to an individual to specifically primean immune response to the target antigen. Moreover, because theinduction of neutralizing antibodies can also be primed withP₃CSS-conjugated epitopes, two such compositions can be combined to moreeffectively elicit both humoral and cell-mediated responses.

[0353] X.C.4. Vaccine Compositions Comprising DC Pulsed with CTL and/orHTL Peptides

[0354] An embodiment of a vaccine composition in accordance with theinvention comprises ex vivo administration of a cocktail ofepitope-bearing peptides to PBMC, or isolated DC therefrom, from thepatient's blood. A pharmaceutical to facilitate harvesting of DC can beused, such as Progenipoietin™ (Pharmacia-Monsanto, St. Louis, Mo.) orGM-CSF/IL-4. After pulsing the DC with peptides and prior to reinfusioninto patients, the DC are washed to remove unbound peptides. In thisembodiment, a vaccine comprises peptide-pulsed DCs which present thepulsed peptide epitopes complexed with HLA molecules on their surfaces.

[0355] The DC can be pulsed ex vivo with a cocktail of peptides, some ofwhich stimulate CTL responses to 108P5H8. Optionally, a helper T cell(HTL) peptide, such as a natural or artificial loosely restricted HLAClass II peptide, can be included to facilitate the CTL response. Thus,a vaccine in accordance with the invention is used to treat a cancerwhich expresses or overexpresses 108P5H8.

[0356] X.D. Adoptive Immunotherapy

[0357] Antigenic 108P5H8-related peptides are used to elicit a CTLand/or HTL response ex vivo, as well. The resulting CTL or HTL cells,can be used to treat tumors in patients that do not respond to otherconventional forms of therapy, or will not respond to a therapeuticvaccine peptide or nucleic acid in accordance with the invention. Exvivo CTL or HTL responses to a particular antigen are induced byincubating in tissue culture the patient's, or genetically compatible,CTL or HTL precursor cells together with a source of antigen-presentingcells (APC), such as dendritic cells, and the appropriate immunogenicpeptide. After an appropriate incubation time (typically about 7-28days), in which the precursor cells are activated and expanded intoeffector cells, the cells are infused back into the patient, where theywill destroy (CTL) or facilitate destruction (HTL) of their specifictarget cell (e.g., a tumor cell). Transfected dendritic cells may alsobe used as antigen presenting cells.

[0358] X.E. Administration of Vaccines for Therapeutic or ProphylacticPurposes

[0359] Pharmaceutical and vaccine compositions of the invention aretypically used to treat and/or prevent a cancer that expresses oroverexpresses 108P5H8. In therapeutic applications, peptide and/ornucleic acid compositions are administered to a patient in an amountsufficient to elicit an effective B cell, CTL and/or HTL response to theantigen and to cure or at least partially arrest or slow symptoms and/orcomplications. An amount adequate to accomplish this is defined as“therapeutically effective dose.” Amounts effective for this use willdepend on, e.g., the particular composition administered, the manner ofadministration, the stage and severity of the disease being treated, theweight and general state of health of the patient, and the judgment ofthe prescribing physician.

[0360] For pharmaceutical compositions, the immunogenic peptides of theinvention, or DNA encoding them, are generally administered to anindividual already bearing a tumor that expresses 108P5H8. The peptidesor DNA encoding them can be administered individually or as fusions ofone or more peptide sequences. Patients can be treated with theimmunogenic peptides separately or in conjunction with other treatments,such as surgery, as appropriate.

[0361] For therapeutic use, administration should generally begin at thefirst diagnosis of 108P5H8-associated cancer. This is followed byboosting doses until at least symptoms are substantially abated and fora period thereafter. The embodiment of the vaccine composition (i.e.,including, but not limited to embodiments such as peptide cocktails,polyepitopic polypeptides, minigenes, or TAA-specific CTLs or pulseddendritic cells) delivered to the patient may vary according to thestage of the disease or the patient's health status. For example, in apatient with a tumor that expresses 108P5H8, a vaccine comprising108P5H8-specific CTL may be more efficacious in killing tumor cells inpatient with advanced disease than alternative embodiments.

[0362] It is generally important to provide an amount of the peptideepitope delivered by a mode of administration sufficient to effectivelystimulate a cytotoxic T cell response; compositions which stimulatehelper T cell responses can also be given in accordance with thisembodiment of the invention.

[0363] The dosage for an initial therapeutic immunization generallyoccurs in a unit dosage range where the lower value is about 1, 5, 50,500, or 1,000 μg and the higher value is about 10,000; 20,000; 30,000;or 50,000 μg. Dosage values for a human typically range from about 500μg to about 50,000 μg per 70 kilogram patient. Boosting dosages ofbetween about 1.0 μg to about 50,000 μg of peptide pursuant to aboosting regimen over weeks to months may be administered depending uponthe patient's response and condition as determined by measuring thespecific activity of CTL and HTL obtained from the patient's blood.Administration should continue until at least clinical symptoms orlaboratory tests indicate that the neoplasia, has been eliminated orreduced and for a period thereafter. The dosages, routes ofadministration, and dose schedules are adjusted in accordance withmethodologies known in the art.

[0364] In certain embodiments, the peptides and compositions of thepresent invention are employed in serious disease states, that is,life-threatening or potentially life threatening situations. In suchcases, as a result of the minimal amounts of extraneous substances andthe relative nontoxic nature of the peptides in preferred compositionsof the invention, it is possible and may be felt desirable by thetreating physician to administer substantial excesses of these peptidecompositions relative to these stated dosage amounts.

[0365] The vaccine compositions of the invention can also be used purelyas prophylactic agents. Generally the dosage for an initial prophylacticimmunization generally occurs in a unit dosage range where the lowervalue is about 1, 5, 50, 500, or 1000 μg and the higher value is about10,000; 20,000; 30,000; or 50,000 μg. Dosage values for a humantypically range from about 500 μg to about 50,000 μg per 70 kilogrampatient. This is followed by boosting dosages of between about 1.0 μg toabout 50,000 μg of peptide administered at defined intervals from aboutfour weeks to six months after the initial administration of vaccine.The immunogenicity of the vaccine can be assessed by measuring thespecific activity of CTL and HTL obtained from a sample of the patient'sblood.

[0366] The pharmaceutical compositions for therapeutic treatment areintended for parenteral, topical, oral, nasal, intrathecal, or local(e.g. as a cream or topical ointment) administration. Preferably, thepharmaceutical compositions are administered parentally, e.g.,intravenously, subcutaneously, intradermally, or intramuscularly. Thus,the invention provides compositions for parenteral administration whichcomprise a solution of the immunogenic peptides dissolved or suspendedin an acceptable carrier, preferably an aqueous carrier.

[0367] A variety of aqueous carriers may be used, e.g., water, bufferedwater, 0.8% saline, 0.3% glycine, hyaluronic acid and the like. Thesecompositions may be sterilized by conventional, well-known sterilizationtechniques, or may be sterile filtered. The resulting aqueous solutionsmay be packaged for use as is, or lyophilized, the lyophilizedpreparation being combined with a sterile solution prior toadministration.

[0368] The compositions may contain pharmaceutically acceptableauxiliary substances as required to approximate physiologicalconditions, such as pH-adjusting and buffering agents, tonicityadjusting agents, wetting agents, preservatives, and the like, forexample, sodium acetate, sodium lactate, sodium chloride, potassiumchloride, calcium chloride, sorbitan monolaurate, triethanolamineoleate, etc.

[0369] The concentration of peptides of the invention in thepharmaceutical formulations can vary widely, i.e., from less than about0.1%, usually at or at least about 2% to as much as 20% to 50% or moreby weight, and will be selected primarily by fluid volumes, viscosities,etc., in accordance with the particular mode of administration selected.

[0370] A human unit dose form of a composition is typically included ina pharmaceutical composition that comprises a human unit dose of anacceptable carrier, in one embodiment an aqueous carrier, and isadministered in a volume/quantity that is known-by those of skill in theart to be used for administration of such compositions to humans (see,e.g., Remington's Pharmaceutical Sciences, 17^(th) Edition, A. Gennaro,Editor, Mack Publishing Co., Easton, Pa., 1985). For example a peptidedose for initial immunization can be from about 1 to about 50,000 μg,generally 100-5,000 μg, for a 70 kg patient. For example, for nucleicacids an initial immunization may be performed using an expressionvector in the form of naked nucleic acid administered IM (or SC or ID)in the amounts of 0.5-5 mg at multiple sites. The nucleic acid (0.1 to1000 μg) can also be administered using a gene gun. Following anincubation period of 3-4 weeks, a booster dose is then administered. Thebooster can be recombinant fowlpox virus administered at a dose of 5-10⁷to 5×10⁹ pfu.

[0371] For antibodies, a treatment generally involves repeatedadministration of the anti-108P5H8 antibody preparation, via anacceptable route of administration such as intravenous injection (IV),typically at a dose in the range of about 0.1 to about 10 mg/kg bodyweight. In general, doses in the range of 10-500 mg mAb per week areeffective and well tolerated. Moreover, an initial loading dose ofapproximately 4 mg/kg patient body weight IV, followed by weekly dosesof about 2 mg/kg IV of the anti-108P5H8 mAb preparation represents anacceptable dosing regimen. As appreciated by those of skill in the art,various factors can influence the ideal dose in a particular case. Suchfactors include, for example, half life of a composition, the bindingaffinity of an Ab, the immunogenicity of a substance, the degree of108P5H8 expression in the patient, the extent of circulating shed108P5H8 antigen, the desired steady-state concentration level, frequencyof treatment, and the influence of chemotherapeutic or other agents usedin combination with the treatment method of the invention, as well asthe health status of a particular patient. Non-limiting preferred humanunit doses are, for example, 500 μg-1 mg, 1 mg-50 mg, 50 mg-100 mg, 100mg-200 mg, 200 mg-300 mg, 400 mg-500 mg, 500 mg-600 mg, 600 mg-700 mg,700 mg-800 mg, 800 mg-900 mg, 900 mg-1 g, or 1 mg-700 mg. In certainembodiments, the dose is in a range of 2-5 mg/kg body weight, e.g., withfollow on weekly doses of 1-3 mg/kg; 0.5 mg, 1, 2, 3, 4, 5, 6, 7, 8, 9,10 mg/kg body weight followed, e.g., in two, three or four weeks byweekly doses; 0.5-10 mg/kg body weight, e.g., followed in two, three orfour weeks by weekly doses; 225, 250, 275, 300, 325, 350, 375, 400 mg m²of body area weekly; 1-600 mg m² of body area weekly; 225-400 mg m² ofbody area weekly; these does can be followed by weekly doses for 2, 3,4, 5, 6, 7, 8, 9, 19, 11, 12 or more weeks.

[0372] In one embodiment, human unit dose forms of polynucleotidescomprise a suitable dosage range or effective amount that provides anytherapeutic effect. As appreciated by one of ordinary skill in the art atherapeutic effect depends on a number of factors, including thesequence of the polynucleotide, molecular weight of the polynucleotideand route of administration. Dosages are generally selected by thephysician or other health care professional in accordance with a varietyof parameters known in the art, such as severity of symptoms, history ofthe patient and the like. Generally, for a polynucleotide of about 20bases, a dosage range may be selected from, for example, anindependently selected lower limit such as about 0.1, 0.25, 0.5, 1, 2,5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400 or 500 mg/kgup to an independently selected upper limit, greater than the lowerlimit, of about 60, 80, 100, 200, 300, 400, 500, 750, 1000, 1500, 2000,3000, 4000, 5000, 6000, 7000, 8000, 9000 or 10,000 mg/kg. For example, adose may be about any of the following: 0.1 to 100 mg/kg, 0.1 to 50mg/kg, 0.1 to 25 mg/kg, 0.1 to 10 mg/kg, 1 to 500 mg/kg, 100 to 400mg/kg, 200 to 300 mg/kg, 1 to 100 mg/kg, 100 to 200 mg/kg, 300 to 400mg/kg, 400 to 500 mg/kg, 500 to 1000 mg/kg, 500 to 5000 mg/kg, or 500 to10,000 mg/kg. Generally, parenteral routes of administration may requirehigher doses of polynucleotide compared to more direct application tothe nucleotide to diseased tissue, as do polynucleotides of increasinglength.

[0373] In one embodiment, human unit dose forms of T-cells comprise asuitable dosage range or effective amount that provides any therapeuticeffect. As appreciated by one of ordinary skill in the art, atherapeutic effect depends on a number of factors. Dosages are generallyselected by the physician or other health care professional inaccordance with a variety of parameters known in the art, such asseverity of symptoms, history of the patient and the like. A dose may beabout 10⁴ cells to about 10⁶ cells, about 10⁶ cells to about 10⁸ cells,about 10⁸ to about 10¹¹ cells, or about 10⁸ to about 5×10¹⁰ cells. Adose may also about 10⁶ cells/m² to about 10¹⁰ cells/m², or about 10⁶cells/m² to about 10⁸ cells/m².

[0374] Proteins(s) of the invention, and/or nucleic acids encoding theprotein(s), can also be administered via liposomes, which may also serveto: 1) target the proteins(s) to a particular tissue, such as lymphoidtissue; 2) to target selectively to diseases cells; or, 3) to increasethe half-life of the peptide composition. Liposomes include emulsions,foams, micelles, insoluble monolayers, liquid crystals, phospholipiddispersions, lamellar layers and the like. In these preparations, thepeptide to be delivered is incorporated as part of a liposome, alone orin conjunction with a molecule which binds to a receptor prevalent amonglymphoid cells, such as monoclonal antibodies which bind to the CD45antigen, or with other therapeutic or immunogenic compositions. Thus,liposomes either filled or decorated with a desired peptide of theinvention can be directed to the site of lymphoid cells, where theliposomes then deliver the peptide compositions. Liposomes for use inaccordance with the invention are formed from standard vesicle-forminglipids, which generally include neutral and negatively chargedphospholipids and a sterol, such as cholesterol. The selection of lipidsis generally guided by consideration of, e.g., liposome size, acidlability and stability of the liposomes in the blood stream. A varietyof methods are available for preparing liposomes, as described in, e.g.,Szoka, et al., Ann. Rev. Biophys. Bioeng. 9:467 (1980), and U.S. Pat.Nos. 4,235,871, 4,501,728, 4,837,028, and 5,019,369.

[0375] For targeting cells of the immune system, a ligand to beincorporated into the liposome can include, e.g., antibodies orfragments thereof specific for cell surface determinants of the desiredimmune system cells. A liposome suspension containing a peptide may beadministered intravenously, locally, topically, etc. in a dose whichvaries according to, inter alia, the manner of administration, thepeptide being delivered, and the stage of the disease being treated.

[0376] For solid compositions, conventional nontoxic solid carriers maybe used which include, for example, pharmaceutical grades of mannitol,lactose, starch, magnesium stearate, sodium saccharin, talcum,cellulose, glucose, sucrose, magnesium carbonate, and the like. For oraladministration, a pharmaceutically acceptable nontoxic composition isformed by incorporating any of the normally employed excipients, such asthose carriers previously listed, and generally 10-95% of activeingredient, that is, one or more peptides of the invention, and morepreferably at a concentration of 25%-75%.

[0377] For aerosol administration, immunogenic peptides are preferablysupplied in finely divided form along with a surfactant and propellant.Typical percentages of peptides are about 0.01%-20% by weight,preferably about 1%-10%. The surfactant must, of course, be nontoxic,and preferably soluble in the propellant. Representative of such agentsare the esters or partial esters of fatty acids containing from about 6to 22 carbon atoms, such as caproic, octanoic, lauric, palmitic,stearic, linoleic, linolenic, olesteric and oleic acids with analiphatic polyhydric alcohol or its cyclic anhydride. Mixed esters, suchas mixed or natural glycerides may be employed. The surfactant mayconstitute about 0.1%-20% by weight of the composition, preferably about0.25-5%. The balance of the composition is ordinarily propellant. Acarrier can also be included, as desired, as with, e.g., lecithin forintranasal delivery.

[0378] XI.) Diagnostic and Prognostic Embodiments of 108P5H8.

[0379] As disclosed herein, 108P5H8 polynucleotides, polypeptides,reactive cytotoxic T cells (CTL), reactive helper T cells (HTL) andanti-polypeptide antibodies are used in well known diagnostic,prognostic and therapeutic assays that examine conditions associatedwith dysregulated cell growth such as cancer, in particular the cancerslisted in Table I (see, e.g., both its specific pattern of tissueexpression as well as its overexpression in certain cancers as describedfor example in Example 4).

[0380] 108P5H8 can be analogized to a prostate associated antigen PSA,the archetypal marker that has been used by medical practitioners foryears to identify and monitor the presence of prostate cancer (see,e.g., Merrill et al., J. Urol. 163(2): 503-5120 (2000); Polascik et al.,J. Urol. August; 162(2):293-306 (1999) and Fortier et al., J. Nat.Cancer Inst. 91(19): 1635-1640(1999)). A variety of other diagnosticmarkers are also used in similar contexts including p53 and K-ras (see,e.g., Tulchinsky et al., Int J Mol Med July 4, 1999(1):99-102 andMinimoto et al., Cancer Detect Prev 2000;24(1):1-12). Therefore, thisdisclosure of 108P5H8 polynucleotides and polypeptides (as well as108P5H8 polynucleotide probes and anti-108P5H8 antibodies used toidentify the presence of these molecules) and their properties allowsskilled artisans to utilize these molecules in methods that areanalogous to those used, for example, in a variety of diagnostic assaysdirected to examining conditions associated with cancer.

[0381] Typical embodiments of diagnostic methods which utilize the108P5H8 polynucleotides, polypeptides, reactive T cells and antibodiesare analogous to those methods from well-established diagnostic assayswhich employ, e.g., PSA polynucleotides, polypeptides, reactive T cellsand antibodies. For example, just as PSA polynucleotides are used asprobes (for example in Northern analysis, see, e.g., Sharief et al.,Biochem. Mol. Biol. Int. 33(3):567-74(1994)) and primers (for example inPCR analysis, see, e.g., Okegawa et al., J. Urol. 163(4): 1189-1190(2000)) to observe the presence and/or the level of PSA mRNAs in methodsof monitoring PSA overexpression or the metastasis of prostate cancers,the 108P5H8 polynucleotides described herein can be utilized in the sameway to detect 108P5H8 overexpression or the metastasis of prostate andother cancers expressing this gene. Alternatively, just as PSApolypeptides are used to generate antibodies specific for PSA which canthen be used to observe the presence and/or the level of PSA proteins inmethods to monitor PSA protein overexpression (see, e.g., Stephan etal., Urology 55(4):560-3 (2000)) or the metastasis of prostate cells(see, e.g., Alanen et al., Pathol. Res. Pract. 192(3):233-7 (1996)), the108P5H8 polypeptides described herein can be utilized to generateanitbodies for use in detecting 108P5H8 overexpression or the metastasisof prostate cells and cells of other cancers expressing this gene.

[0382] Specifically, because metastases involves the movement of cancercells from an organ of origin (such as the lung or prostate gland etc.)to a different area of the body (such as a lymph node), assays whichexamine a biological sample for the presence of cells expressing 108P5H8polynucleotides and/or polypeptides can be used to provide evidence ofmetastasis. For example, when a biological sample from tissue that doesnot normally contain 108P5H8-expressing cells (lymph node) is found tocontain 108P5H8-expressing cells such as the 108P5H8 expression seen inLAPC4 and LAPC9, xenografts isolated from lymph node and bonemetastasis, respectively, this finding is indicative of metastasis.

[0383] Alternatively 108P5H8 polynucleotides and/or polypeptides can beused to provide evidence of cancer, for example, when cells in abiological sample that do not normally express 108P5H8 or express108P5H8 at a different level are found to express 108P5H8 or have anincreased expression of 108P5H8 (see, e.g., the 108P5H8 expression inthe cancers listed in Table I and in patient samples etc. shown in theaccompanying Figures). In such assays, artisans may further wish togenerate supplementary evidence of metastasis by testing the biologicalsample for the presence of a second tissue restricted marker (inaddition to 108P5H8) such as PSA, PSCA etc. (see, e.g.; Alanen et al.,Pathol. Res. Pract. 192(3): 233-237 (1996)).

[0384] Just as PSA polynucleotide fragments and polynucleotide variantsare employed by skilled artisans for use in methods of monitoring PSA,108P5H8 polynucleotide fragments and polynucleotide variants are used inan analogous manner. In particular, typical PSA polynucleotides used inmethods of monitoring PSA are probes or primers which consist offragments of the PSA cDNA sequence. Illustrating this, primers used toPCR amplify a PSA polynucleotide must include less than the whole PSAsequence to function in the polymerase chain reaction. In the context ofsuch PCR reactions, skilled artisans generally create a variety ofdifferent polynucleotide fragments that can be used as primers in orderto amplify different portions of a polynucleotide of interest or tooptimize amplification reactions (see, e.g., Caetano-Anolles, G.Biotechniques 25(3): 472-476, 478-480 (1998); Robertson et al., MethodsMol. Biol. 98:121-154 (1998)). An additional illustration of the use ofsuch fragments is provided in Example 4, where a 108P5H8 polynucleotidefragment is used as a probe to show the expression of 108P5H8 RNAs incancer cells. In addition, variant polynucleotide sequences aretypically used as primers and probes for the corresponding mRNAs in PCRand Northern analyses (see, e.g., Sawai et al., Fetal Diagn. Ther.November 1996-December 11(6):407-13 and Current Protocols In MolecularBiology, Volume 2, Unit 2, Frederick M. Ausubel et al. eds., 1995)).Polynucleotide fragments and variants are useful in this context wherethey are capable of binding to a target polynucleotide sequence (e.g., a108P5H8 polynucleotide shown in FIG. 2 or variant thereof) underconditions of high stringency.

[0385] Furthermore, PSA polypeptides which contain an epitope that canbe recognized by an antibody or T cell that specifically binds to thatepitope are used in methods of monitoring PSA. 108P5H8 polypeptidefragments and polypeptide analogs or variants can also be used in ananalogous manner. This practice of using polypeptide fragments orpolypeptide variants to generate antibodies (such as anti-PSA antibodiesor T cells) is typical in the art with a wide variety of systems such asfusion proteins being used by practitioners (see, e.g., CurrentProtocols In Molecular Biology, Volume 2, Unit 16, Frederick M. Ausubelet al. eds., 1995). In this context, each epitope(s) functions toprovide the architecture with which an antibody or T cell is reactive.Typically, skilled artisans create a variety of different polypeptidefragments that can be used in order to generate immune responsesspecific for different portions of a polypeptide of interest (see, e.g.,U.S. Pat. No. 5,840,501 and U.S. Pat. No. 5,939,533). For example it maybe preferable to utilize a polypeptide comprising one of the 108P5H8biological motifs discussed herein or a motif-bearing subsequence whichis readily identified by one of skill in the art based on motifsavailable in the art. Polypeptide fragments, variants or analogs aretypically useful in this context as long as they comprise an epitopecapable of generating an antibody or T cell specific for a targetpolypeptide sequence (e.g. a 108P5H8 polypeptide shown in FIG. 3).

[0386] As shown herein, the 108P5H8 polynucleotides and polypeptides (aswell as the 108P5H8 polynucleotide probes and anti-108P5H8 antibodies orT cells used to identify the presence of these molecules) exhibitspecific properties that make them useful in diagnosing cancers such asthose listed in Table I. Diagnostic assays that measure the presence of108P5H8 gene products, in order to evaluate the presence or onset of adisease condition described herein, such as prostate cancer, are used toidentify patients for preventive measures or further monitoring, as hasbeen done so successfully with PSA. Moreover, these materials satisfy aneed in the art for molecules having similar or complementarycharacteristics to PSA in situations where, for example, a definitediagnosis of metastasis of prostatic origin cannot be made on the basisof a test for PSA alone (see, e.g., Alanen et al., Pathol. Res. Pract.192(3): 233-237 (1996)), and consequently, materials such as 108P5H8polynucleotides and polypeptides (as well as the 108P5H8 polynucleotideprobes and anti-108P5H8 antibodies used to identify the presence ofthese molecules) need to be employed to confirm a metastases ofprostatic origin.

[0387] Finally, in addition to their use in diagnostic assays, the108P5H8 polynucleotides disclosed herein have a number of otherutilities such as their use in the identification of oncogeneticassociated chromosomal abnormalities in the chromosomal region to whichthe 108P5H8 gene maps (see Example 3 below). Moreover, in addition totheir use in diagnostic assays, the 108P5H8-related proteins andpolynucleotides disclosed herein have other utilities such as their usein the forensic analysis of tissues of unknown origin (see, e.g.,Takahama K Forensic Sci Int Jun. 28, 1996;80(1-2): 63-9).

[0388] Additionally, 108P5H8-related proteins or polynucleotides of theinvention can be used to treat a pathologic condition characterized bythe over-expression of 108P5H8. For example, the amino acid or nucleicacid sequence of FIG. 2 or FIG. 3, or fragments of either, can be usedto generate an immune response to a 108P5H8 antigen. Antibodies or othermolecules that react with 108P5H8 can be used to modulate the functionof this molecule, and thereby provide a therapeutic benefit.

[0389] XII.) Inhibition of 108P5H8 Protein Function

[0390] The invention includes various methods and compositions forinhibiting the binding of 108P5H8 to its binding partner or itsassociation with other protein(s) as well as methods for inhibiting108P5H8 function.

[0391] XII.A.) Inhibition of 108P5H8 with Intracellular Antibodies

[0392] In one approach, a recombinant vector that encodes single chainantibodies that specifically bind to 108P5H8 are introduced into 108P5H8expressing cells via gene transfer technologies. Accordingly, theencoded single chain anti-108P5H8 antibody is expressed intracellularly,binds to 108P5H8 protein, and thereby inhibits its function. Methods forengineering such intracellular single chain antibodies are well known.Such intracellular antibodies, also known as “intrabodies”, arespecifically targeted to a particular compartment within the cell,providing control over where the inhibitory activity of the treatment isfocused. This technology has been successfully applied in the art (forreview, see Richardson and Marasco, 1995, TIBTECH vol. 13). Intrabodieshave been shown to virtually eliminate the expression of otherwiseabundant cell surface receptors (see, e.g., Richardson et al., 1995,Proc. Natl. Acad. Sci. USA 92: 3137-3141; Beerli et al., 1994, J. Biol.Chem. 289: 23931-23936; Deshane et al., 1994, Gene Ther. 1: 332-337).

[0393] Single chain antibodies comprise the variable domains of theheavy and light chain joined by a flexible linker polypeptide, and areexpressed as a single polypeptide. Optionally, single chain antibodiesare expressed as a single chain variable region fragment joined to thelight chain constant region. Well-known intracellular traffickingsignals are engineered into recombinant polynucleotide vectors encodingsuch single chain antibodies in order to precisely target the intrabodyto the desired intracellular compartment. For example, intrabodiestargeted to the endoplasmic reticulum (ER) are engineered to incorporatea leader peptide and, optionally, a C-terminal ER retention signal, suchas the KDEL amino acid motif. Intrabodies intended to exert activity inthe nucleus are engineered to include a nuclear localization signal.Lipid moieties are joined to intrabodies in order to tether theintrabody to the cytosolic side of the plasma membrane. Intrabodies canalso be targeted to exert function in the cytosol. For example,cytosolic intrabodies are used to sequester factors within the cytosol,thereby preventing them from being transported to their natural cellulardestination.

[0394] In one embodiment, intrabodies are used to capture 108P5H8 in thenucleus, thereby preventing its activity within the nucleus. Nucleartargeting signals are engineered into such 108P5H8 intrabodies in orderto achieve the desired targeting. Such 108P5H8 intrabodies are designedto bind specifically to a particular 108P5H8 domain. In anotherembodiment, cytosolic intrabodies that specifically bind to a 108P5H8protein are used to prevent 108P5H8 from gaining access to the nucleus,thereby preventing it from exerting any biological activity within thenucleus (e.g., preventing 108P5H8 from forming transcription complexeswith other factors).

[0395] In order to specifically direct the expression of suchintrabodies to particular cells, the transcription of the intrabody isplaced under the regulatory control of an appropriate tumor-specificpromoter and/or enhancer. In order to target intrabody expressionspecifically to prostate, for example, the PSA promoter and/orpromoter/enhancer can be utilized (See, for example, U.S. Pat. No.5,919,652 issued Jul. 6, 1999).

[0396] XII.B.) Inhibition of 108P5H8 with Recombinant Proteins

[0397] In another approach, recombinant molecules bind to 108P5H8 andthereby inhibit 108P5H8 function. For example, these recombinantmolecules prevent or inhibit 108P5H8 from accessing/binding to itsbinding partner(s) or associating with other protein(s). Suchrecombinant molecules can, for example, contain the reactive part(s) ofa 108P5H8 specific antibody molecule. In a particular embodiment, the108P5H8 binding domain of a 108P5H8 binding partner is engineered into adimeric fusion protein, whereby the fusion protein comprises two 108P5H8ligand binding domains linked to the Fc portion of a human IgG, such ashuman IgG1. Such IgG portion can contain, for example, the C_(H)2 andC_(H)3 domains and the hinge region, but not the C_(H)1 domain. Suchdimeric fusion proteins are administered in soluble form to patientssuffering from a cancer associated with the expression of 108P5H8,whereby the dimeric fusion protein specifically binds to 108P5H8 andblocks 108P5H8 interaction with a binding partner. Such dimeric fusionproteins are further combined into multimeric proteins using knownantibody linking technologies.

[0398] XII.C.) Inhibition of 108P5H8 Transcription or Translation

[0399] The present invention also comprises various methods andcompositions for inhibiting the transcription of the 108P5H8 gene.Similarly, the invention also provides methods and compositions forinhibiting the translation of 108P5H8 mRNA into protein.

[0400] In one approach, a method of inhibiting the transcription of the108P5H8 gene comprises contacting the 108P5H8 gene with a 108P5H8antisense polynucleotide. In another approach, a method of inhibiting108P5H8 mRNA translation comprises contacting a 108P5H8 mRNA with anantisense polynucleotide. In another approach, a 108P5H8 specificribozyme is used to cleave a 108P5H8 message, thereby inhibitingtranslation. Such antisense and ribozyme based methods can also bedirected to the regulatory regions of the 108P5H8 gene, such as 108P5H8promoter and/or enhancer elements. Similarly, proteins capable ofinhibiting a 108P5H8 gene transcription factor are used to inhibit108P5H8 mRNA transcription. The various polynucleotides and compositionsuseful in the aforementioned methods have been described above. The useof antisense and ribozyme molecules to inhibit transcription andtranslation is well known in the art.

[0401] Other factors that inhibit the transcription of 108P5H8 byinterfering with 108P5H8 transcriptional activation are also useful totreat cancers expressing 108P5H8. Similarly, factors that interfere with108P5H8 processing are useful to treat cancers that express 108P5H8.Cancer treatment methods utilizing such factors are also within thescope of the invention.

[0402] XII.D.) General Considerations for Therapeutic Strategies

[0403] Gene transfer and gene therapy technologies can be used todeliver therapeutic polynucleotide molecules to tumor cells synthesizing108P5H8 (i.e., antisense, ribozyme, polynucleotides encoding intrabodiesand other 108P5H8 inhibitory molecules). A number of gene therapyapproaches are known in the art. Recombinant vectors encoding 108P5H8antisense polynucleotides, ribozymes, factors capable of interferingwith 108P5H8 transcription, and so forth, can be delivered to targettumor cells using such gene therapy approaches.

[0404] The above therapeutic approaches can be combined with any one ofa wide variety of surgical, chemotherapy or radiation therapy regimens.The therapeutic approaches of the invention can enable the use ofreduced dosages of chemotherapy (or other therapies) and/or lessfrequent administration, an advantage for all patients and particularlyfor those that do not tolerate the toxicity of the chemotherapeuticagent well.

[0405] The anti-tumor activity of a particular composition (e.g.,antisense, ribozyme, intrabody), or a combination of such compositions,can be evaluated using various in vitro and in vivo assay systems. Invitro assays that evaluate therapeutic activity include cell growthassays, soft agar assays and other assays indicative of tumor promotingactivity, binding assays capable of determining the extent to which atherapeutic composition will inhibit the binding of 108P5H8 to a bindingpartner, etc.

[0406] In vivo, the effect of a 108P5H8 therapeutic composition can beevaluated in a suitable animal model. For example, xenogenic prostatecancer models can be used, wherein human prostate cancer explants orpassaged xenograft tissues are introduced into immune compromisedanimals, such as nude or SCID mice (Klein et al., 1997, Nature Medicine3: 402-408). For example, PCT Patent Application WO98/16628 and U.S.Pat. No. 6,167,540 describe various xenograft models of human prostatecancer capable of recapitulating the development of primary tumors,micrometastasis, and the formation of osteoblastic metastasescharacteristic of late stage disease. Efficacy can be predicted usingassays that measure inhibition of tumor formation, tumor regression ormetastasis, and the like.

[0407] In vivo assays that evaluate the promotion of apoptosis areuseful in evaluating therapeutic compositions. In one embodiment,xenografts from tumor bearing mice treated with the therapeuticcomposition can be examined for the presence of apoptotic foci andcompared to untreated control xenograft-bearing mice. The extent towhich apoptotic foci are found in the tumors of the treated miceprovides an indication of the therapeutic efficacy of the composition.

[0408] The therapeutic compositions used in the practice of theforegoing methods can be formulated into pharmaceutical compositionscomprising a carrier suitable for the desired delivery method. Suitablecarriers include any material that when combined with the therapeuticcomposition retains the anti-tumor function of the therapeuticcomposition and is generally non-reactive with the patient's immunesystem. Examples include, but are not limited to, any of a number ofstandard pharmaceutical carriers such as sterile phosphate bufferedsaline solutions, bacteriostatic water, and the like (see, generally,Remington's Pharmaceutical Sciences 16^(th) Edition, A. Osal., Ed.,1980).

[0409] Therapeutic formulations can be solubilized and administered viaany route capable of delivering the therapeutic composition to the tumorsite. Potentially effective routes of administration include, but arenot limited to, intravenous, parenteral, intraperitoneal, intramuscular,intratumor, intradermal, intraorgan, orthotopic, and the like. Apreferred formulation for intravenous injection comprises thetherapeutic composition in a solution of preserved bacteriostatic water,sterile unpreserved water, and/or diluted in polyvinylchloride orpolyethylene bags containing 0.9% sterile Sodium Chloride for Injection,USP. Therapeutic protein preparations can be lyophilized and stored assterile powders, preferably under vacuum, and then reconstituted inbacteriostatic water (containing for example, benzyl alcoholpreservative) or in sterile water prior to injection.

[0410] Dosages and administration protocols for the treatment of cancersusing the foregoing methods will vary with the method and the targetcancer, and will generally depend on a number of other factorsappreciated in the art.

[0411] XIII.) Kits

[0412] For use in the diagnostic and therapeutic applications describedherein, kits are also within the scope of the invention. Such kits cancomprise a carrier, package or container that is compartmentalized toreceive one or more containers such as vials, tubes, and the like, eachof the container(s) comprising one of the separate elements to be usedin the method. For example, the container(s) can comprise a probe thatis or can be detectably labeled. Such probe can be an antibody orpolynucleotide specific for a 108P5H8-related protein or a 108P5H8 geneor message, respectively. Where the method utilizes nucleic acidhybridization to detect the target nucleic acid, the kit can also havecontainers containing nucleotide(s) for amplification of the targetnucleic acid sequence and/or a container comprising a reporter-means,such as a biotin-binding protein, such as avidin or streptavidin, boundto a reporter molecule, such as an enzymatic, florescent, orradioisotope label. The kit can include all or part of the amino acidsequence of FIG. 2 or FIG. 3 or analogs thereof, or a nucleic acidmolecules that encodes such amino acid sequences.

[0413] The kit of the invention will typically comprise the containerdescribed above and one or more other containers comprising materialsdesirable from a commercial and user standpoint, including buffers,diluents, filters, needles, syringes, and package inserts withinstructions for use.

[0414] A label can be present on the container to indicate that thecomposition is used for a specific therapy or non-therapeuticapplication, and can also indicate directions for either in vivo or invitro use, such as those described above. Directions and or otherinformation can also be included on an insert which is included with thekit.

EXAMPLES

[0415] Various aspects of the invention are further described andillustrated by way of the several examples that follow, none of whichare intended to limit the scope of the invention.

Example 1

[0416] SSH-Generated Isolation of a cDNA Fragment of the 108P5H8 Gene

[0417] To isolate genes that are androgen regulated, theandrogen-dependent prostate cancer cell line LNCaP was grown in mediacontaining charcoal-stripped serum (steroid hormone depleted) for oneweek. The cells were subsequently stimulated with 10 nM mibolerone(synthetic androgen) for 9 h and were harvested as a source of mRNA. The108P5H8 SSH cDNA sequence was derived from a subtraction consisting ofLNCaP cells grown in presence of mibolerone minus LNCaP cells grown inabsence of mibolerone.

[0418] The 108P5H8 SSH cDNA sequence of 448 bp (FIG. 1), showed homologyonly to ESTs in the dbEST database. The full length 108P5H8 cDNAs andORFs are described in FIG. 2 with the protein sequences listed in FIG.3.

[0419] Materials and Methods

[0420] RNA Isolation:

[0421] Tumor tissues were homogenized in Trizol reagent (LifeTechnologies, Gibco BRL) using 10 ml/g tissue or 10 ml/10⁸ cells toisolate total RNA. Poly A RNA was purified from total RNA using Qiagen'sOligotex mRNA Mini and Midi kits. Total and mRNA were quantified byspectrophotometric analysis (O.D. 260/280 nm) and analyzed by gelelectrophoresis.

[0422] Oligonucleotides:

[0423] The following HPLC purified oligonucleotides were used. DPNCDN(cDNA synthesis primer): (SEQ ID NO: 2586) 5′TTTTGATCAAGCTT₃₀3′ Adaptor1: (SEQ ID NO: 2587) 5′CTAATACGACTCACTATAGGGCTCGAGCGGCCGCCCGGGCAG3′ (SEQID NO: 2588) 3′GGCCCGTCCTAG5′ Adaptor 2: (SEQ ID NO: 2589)5′GTAATACGACTCACTATAGGGCAGCGTGGTCGCGGCCGAG3′ (SEQ ID NO: 2590)3′CGGCTCCTAG5′ PCR primer 1: (SEQ ID NO: 2591)5′CTAATACGACTCACTATAGGGC3′ Nested primer (NP) 1: (SEQ ID NO: 2592)5′TCGAGCGGCCGCCCGGGCAGGA3′ Nested primer (NP) 2: (SEQ ID NO: 2593)5′AGCGTGGTCGCGGCCGAGGA3′

[0424] Suppression Subtractive Hybridization:

[0425] Suppression Subtractive Hybridization (SSH) was used to identifycDNAs corresponding to genes that are androgen regulated. The SSHreaction utilized cDNA from LNCaP prostate cancer cells grown inpresence of mibolerone minus LNCaP cells grown in absence of mibolerone.

[0426] The cDNA derived from LNCaP prostate cancer cells grown inabsence of mibolerone was used as the source of the “driver” cDNA, whilethe LNCaP prostate cancer cells grown in presence of mibolerone was usedas the source of the “tester” cDNA. Double stranded cDNAs correspondingto tester and driver cDNAs were synthesized from 2 μg of poly(A)⁺ RNAisolated from the relevant tissue, as described above, using CLONTECH'sPCR-Select cDNA Subtraction Kit and 1 ng of oligonucleotide DPNCDN asprimer. First- and second-strand synthesis were carried out as describedin the Kit's user manual protocol (CLONTECH Protocol No. PT1117-1,Catalog No. K1804-1). The resulting cDNA was digested with Dpn II for 3hrs at 37° C. Digested cDNA was extracted with phenol/chloroform (1:1)and ethanol precipitated.

[0427] Tester cDNA was generated by diluting 1 μl of Dpn II digestedcDNA from the relevant tissue source (see above) (400 ng) in 5 μl ofwater. The diluted cDNA (2 μl, 160 ng) was then ligated to 2 μl ofAdaptor 1 and Adaptor 2 (10 μM), in separate ligation reactions, in atotal volume of 10 μl at 16° C. overnight, using 400 u of T4 DNA ligase(CLONTECH). Ligation was terminated with 1 μl of 0.2 M EDTA and heatingat 72° C. for 5 min.

[0428] The first hybridization was performed by adding 1.5 μl (600 ng)of driver cDNA to each of two tubes containing 1.5 μl (20 ng) Adaptor 1-and Adaptor 2-ligated tester cDNA. In a final volume of 4 μl, thesamples were overlaid with mineral oil, denatured in an MJ Researchthermal cycler at 98° C. for 1.5 minutes, and then were allowed tohybridize for 8 hrs at 68° C. The two hybridizations were then mixedtogether with an additional 1 μl of fresh denatured driver cDNA and wereallowed to hybridize overnight at 68° C. The second hybridization wasthen diluted in 200 μl of 20 mM Hepes, pH 8.3, 50 mM NaCl, 0.2 mM EDTA,heated at 70° C. for 7 min. and stored at −20° C.

[0429] PCR Amplification, Cloning and Sequencing of Gene FragmentsGenerated from SSH:

[0430] To amplify gene fragments resulting from SSH reactions, two PCRamplifications were performed. In the primary PCR reaction 1 μl of thediluted final hybridization mix was added to 1 μl of PCR primer 1 (10μM), 0.5 μl dNTP mix (10 μM), 2.5 μl 10× reaction buffer (CLONTECH) and0.5 μl 50× Advantage cDNA polymerase Mix (CLONTECH) in a final volume of25 μl. PCR 1 was conducted using the following conditions: 75° C. for 5min., 94° C. for 25 sec., then 27 cycles of 94° C. for 10 sec, 66° C.for 30 sec, 72° C. for 1.5 min. Five separate-primary PCR reactions wereperformed for each experiment. The products were pooled and diluted 1:10with water. For the secondary PCR reaction, 1 μl from the pooled anddiluted primary PCR reaction was added to the same reaction mix as usedfor PCR 1, except that primers NP1 and NP2 (10 μM) were used instead ofPCR primer 1. PCR 2 was performed using 10- 12 cycles of 94° C. for 10sec, 68° C. for 30 sec, and 72° C. for 1.5 minutes. The PCR productswere analyzed using 2% agarose gel electrophoresis.

[0431] The PCR products were inserted into pCR2.1 using the T/A vectorcloning kit (Invitrogen). Transformed E. coli were subjected toblue/white and ampicillin selection. White colonies were picked andarrayed into 96 well plates and were grown in liquid culture overnight.To identify inserts, PCR amplification was performed on 1 ml ofbacterial culture using the conditions of PCR1 and NP1 and NP2 asprimers. PCR products were analyzed using 2% agarose gelelectrophoresis.

[0432] Bacterial clones were stored in 20% glycerol in a 96 well format.Plasmid DNA was prepared, sequenced, and subjected to nucleic acidhomology searches of the GenBank, dbest, and NCI-CGAP databases.

[0433] RT-PCR Expression Analysis:

[0434] First strand cDNAs can be generated from 1 μg of mRNA with oligo(dT)12-18 priming using the Gibco-BRL Superscript Preamplificationsystem. The manufacturer's protocol was used which included anincubation for 50 min at 42° C. with reverse transcriptase followed byRNAse H treatment at 37° C. for 20 min. After completing the reaction,the volume can be increased to 200 μl with water prior to normalization.First strand cDNAs from 16 different normal human tissues can beobtained from Clontech.

[0435] Normalization of the first strand cDNAs from multiple tissues wasperformed by using the primers 5′-atatcgccgcgctcgtcgtcgacaa-3′ (SEQ IDNO: 2594) and 5′-agccacacgcagctcattgtagaagg-3′ (SEQ ID NO: 2595) toamplify β-actin. First strand cDNA (5 μl) were amplified in a totalvolume of 50 μl containing 0.4 μM primers, 0.2 μM each dNTPs, 1×PCRbuffer (Clontech, 10 mM Tris-HCL, 1.5 mM MgCl₂, 50 mM KCl, pH8.3) and 1×Klentaq DNA polymerase (Clontech). Five μl of the PCR reaction can beremoved at 18, 20, and 22 cycles and used for agarose gelelectrophoresis. PCR was performed using an MJ Research thermal cyclerunder the following conditions: Initial denaturation can be at 94° C.for 15 sec, followed by a 18, 20, and 22 cycles of 94° C. for 15, 65° C.for 2 min, 72° C. for 5 sec. A final extension at 72° C. was carried outfor 2 min. After agarose gel electrophoresis, the band intensities ofthe 283 bp β-actin bands from multiple tissues were compared by visualinspection. Dilution factors for the first strand cDNAs were calculatedto result in equal β-actin band intensities in all tissues after 22cycles of PCR. Three rounds of normalization can be required to achieveequal band intensities in all tissues after 22 cycles of PCR.

[0436] To determine expression levels of the 108P5H8 gene, 5 μl ofnormalized first strand cDNA were analyzed by PCR using 26, and 30cycles of amplification. Semi-quantitative expression analysis can beachieved by comparing the PCR products at cycle numbers that give lightband intensities.

[0437] A typical RT-PCR expression analysis is shown in FIG. 10. RT-PCRexpression analysis was performed on first strand cDNAs generated usingpools of tissues from multiple samples. The cDNAs were shown to benormalized using beta-actin PCR. Strong expression of 108P5H8 wasobserved in prostate cancer xenograft pool, prostate cancer pool and inthe 2 different prostate cancer metastasis samples. Lower expression wasdetected in bladder cancer pool, kidney cancer pool, colon cancer pool,lung cancer pool, ovary cancer pool, breast cancer pool, metastasispool, pancreas cancer pool, VP1 and VP2.

Example 2

[0438] Full Length Cloning of 108P5H8

[0439] To isolate genes that are androgen regulated, theandrogen-dependent prostate cancer cell line LNCaP was grown in mediacontaining 2% charcoal-stripped serum (steroid hormone depleted) for oneweek. The cells were then stimulated with 10 nM Mibolerone (syntheticandrogen) for 9 hours and were harvested for RNA.

[0440] The gene 108P5H8 was derived from an experiment where cDNAderived from LNCaP cells that was androgen-deprived (by growing in thepresence of charcoal-stripped serum) was subtracted from cDNA derivedfrom LNCaP cells that were stimulated with mibolerone for 9 hours. TheSSH DNA sequence of 448 bp (FIG. 1) is novel and only exhibited homologyto human EST sequences in the dbest database.

[0441] A full length 108P5H8 cDNA clone (108P5H8 v.1) of 2364 base pairs(bp) was cloned from a prostate library (Lambda ZAP Express, Stratagene)(FIG. 2). The cDNA encodes a putative open reading frame (ORF) of 429amino acids.

[0442] 108P5H8 variant 2 and variant 3 were identified. The nucleic acidand protein sequences of all 3 variants are presented in FIG. 3 and FIG.4. The alignments of all 3 108P5H8 variants are presented in FIG. 4. Thenucleic acid sequences of variants 1 and 2 encode identical protein.108P5H8 v.3 has a base pair variation with a C at position 342 of v.1converted into G in v.3. This nucleotide change converted amino acidposition 30 from aspartic acid in the 108P5H8 v.1 and v.2 proteinsequence, to glutamic acid in 108P5H8 v.3.

[0443] Analysis of 108P5H8 protein sequence using the PSORT program(http://psort.nibb.acjp:8800/form.html) reveals 6 predictedtransmembrane domains. Sequence analysis of 108P5H8 reveals homology tothe human zinc transporter protein ZnT4 (Huang and Gitschier, 1997,Nature Genetics 17:292). The 108P5H8 v.1 sequence includes novel 5′ UTRand 3′ UTR sequences, and the molecule contains 75% GC sequence,indicating possible translational regulatory sites.

[0444] To further confirm the parameters of a variant, a variety oftechniques are available in the art, such as full-length cloning,proteomic validation, PCR-based validation, and 5′ RACE validation, etc.(see e.g., Proteomic Validation: Brennan, S. O., et al., Albumin bankspeninsula: a new termination variant characterized by electrospray massspectrometry, Biochem Biophys Acta. Aug. 17, 1999;1433(1-2):321-6;Ferranti P, et al., Differential splicing of pre-messenger RNA producesmultiple forms of mature caprine alpha(s1)-casein, Eur J Biochem. Oct.1, 1997;249(1):1-7. For PCR-based Validation: Wellmann S, et al.,Specific reverse transcription-PCR quantification of vascularendothelial growth factor (VEGF) splice variants by LightCyclertechnology, Clin Chem. April 2001;47(4):654-60; Jia, H. P., et al.,Discovery of new human beta-defensins using a genomics-based approach,Gene. Jan. 24, 2001; 263(1-2):211-8. For PCR-based and 5′ RACEValidation: Brigle, K. E., et al., Organization of the murine reducedfolate carrier gene and identification of variant splice forms, BiochemBiophys Acta. Aug. 7, 1997;1353(2): 191-8).

[0445] It is known in the art that genomic regions are modulated incancers. When the genomic region to which 108P5H8 maps is modulated in aparticular cancer, the variants of 108P5H8 are modulated as well.Disclosed herein is that 108P5H8 has a particular expression profile.Variants of 108P5H8 that are structurally and/or functionally similar to108P5H8 share this expression pattern, thus serving as tumor-associatedmarkers/antigens.

Example 3

[0446] Chromosomal Localization

[0447] Chromosomal localization can implicate genes in diseasepathogenesis. Several chromosome mapping approaches are available,including fluorescent in situ hybridization (FISH), human/hamsterGenebridge4 radiation hybrid (RH) panels (Walter et al., 1994; NatureGenetics 7:22; Research Genetics, Huntsville Ala.), human-rodent somaticcell hybrid panels such as is available from the Coriell Institute(Camden, N.J.), and genomic viewers utilizing BLAST homologies tosequenced and mapped genomic clones (NCBI, Bethesda, Md.).

[0448] The chromosomal localization of 108P5H8 using the GeneBridge4radiation hybrid panel was performed using the the following PCRprimers: (SEQ. ID. No.: 2596) 108P5H8.1 5′ TGCACACTGGACTTCGTAGAGTAA 3′(SEQ. ID. No.: 2597) 108P5H8.2 5′ AAAGCTGTGAGAGTGGCTGAGAAA 3′

[0449] The resulting mapping vector for the 93 radiation hybrid panelDNAs was:100101001101000101000000000000110100000001201100001011000100001011100010100010000020110110121

[0450] This mapping vector and the mapping program athttp://www-genome.wi.mit.edu/cgi-bin/contig/rhmapper.p1 placed 108P5H8to chromosome 15q15.2-q21.1.

Example 4

[0451] Expression Analysis of 108P5H8 in Normal Tissues and PatientSpecimens

[0452] Expression of 108P5H8 was analyzed by RT-PCR (FIG. 10). Firststrand cDNA was prepared from vital pool 1 (VP1: liver, lung andkidney), vital pool 2 (VP2, pancreas, colon and stomach), prostatexenograft pool (LAPC-4AD, LAPC-4AI, LAPC-9AD, LAPC-9AI), prostate cancerpool, bladder cancer pool, kidney cancer pool, colon cancer pool, lungcancer pool, ovary cancer pool, breast cancer pool, metastasis cancerpool, pancreas cancer pool, and from prostate cancer metastasis to lymphnode from two different patients. Normalization was performed by PCRusing primers to actin and GAPDH. Semi-quantitative PCR, using primersto 108P5H8, was performed at 26 and 30 cycles of amplification. Strongexpression of 108P5H8 was observed in prostate cancer xenograft pool,prostate cancer pool and in the 2 different prostate cancer metastasissamples. Lower expression was detected in bladder cancer pool, kidneycancer pool, colon cancer pool, lung cancer pool, ovary cancer pool,breast cancer pool, metastasis pool, pancreas cancer pool, VP1 and VP2.

[0453] Extensive Northern blot analysis of 108P5H8 in 16 human normaltissues confirmed the expression observed by RT-PCR (FIG. 11). Anapproximately 7 kb 108P5H8 transcript was strongly expressed inprostate. Significantly lower expression was detected in other tissues.

[0454]FIG. 11C shows expression of 108P5H8 in prostate cancerxenografts. RNA was extracted from prostate cancer xenografts, LAPC-4AD,LAPC-4AI, LAPC-9AD, and LAPC-9AI. Northern blot with 10 μg of totalRNA/lane was probed with 108P5H8 SSH sequence. Results showed expressionof 108P5H8 in all four xenograft tissues. More detailed analysis of thexenografts shows that 108P5H8is highly expressed in the xenografts evenwhen grown within the tibia of mice (FIG. 12). The expression isincreased when the LAPC-4 xenograft is grown within a human bone implant(LAPC-4 AD²) It is possible that the human bone environment increasesand/or induces the expression of 108P5H8. Northern blot analysis alsoshowed that 108P5H8 is expressed in all human cancer cell lines testedsuch as prostate, bladder, brain, lung, kidney, breast, testis and ovarycancer cell lines (FIG. 13).

[0455] Expression of 108P5H8 was assayed in a panel of human cancers (T)and their respective matched normal tissues (N) on RNA dot blots (FIG.14). 108P5H8 expression was detected in prostate, kidney, uterus andstomach cancers. The expression detected in some normal adjacent tissues(isolated from diseased tissues), but not in normal tissues (isolatedfrom healthy donors), may indicate that these tissues are not fullynormal and that 108P5H8 may be expressed in early stage tumors. 108P5H8was also expressed in all 9 human cancer cell lines tested.

[0456] To test expression of 108P5H8 in patient cancer specimens, RNAwas extracted from prostate cancer tumors (T) and their matched normaladjacent tissue (N_(AT)). Northern blots with 10 μg of total RNA/lanewere probed with 108P5H8 SSH sequence (FIG. 15). Results showedexpression of 108P5H8 in all prostate patient specimens tested.

[0457] 108P5H8 was isolated from an experiment where cDNA derived fromLNCaP cells that was androgen-deprived (by growing in the presence ofcharcoal-stripped serum) was subtracted from cDNA derived from LNCaPcells that were stimulated with mibolerone. To assess whether 108P5H8 isandrogen-regulated, LNCaP cells were grown in charcoal-stripped mediumand stimulated with the synthetic androgen mibolerone, for either 14 or24 hours (FIG. 16). Northern blots with 10 μg of total RNA/lane wereprobed with either the 108P5H8 sequence (FIG. 16A). Results showexpression of 108P5H8 is not regulated by androgen. The experimentalsamples were confirmed by testing for the expression of theandrogen-regulated prostate cancer gene PSA (FIG. 16B). This experimentshows that, as expected, PSA levels go down in presence ofcharcoal-stripped serum, and expression is induced at 14 and 24 hours inpresence of the synthetic androgen. A picture of the ethidium-bromidestaining of the RNA gel is also presented (FIG. 16C).

[0458]FIG. 17 shows expression of 108P5H8 in cancer metastasis patientspecimens. RNA was extracted from prostate cancer metastasis to lymphnode isolated from 2 different patients, as well as from normal bladder(NB), normal kidney (NK), normal lung (NL), normal breast (NBr), normalovary (NO), and normal pancreas (NPa). Northern blots with 10 μg oftotal RNA/lane was probed with 108P5H8 sequence. The results showexpression of 108P5H8 in both cancer metastasis samples but not innormal tissues.

[0459] 108P5H8 expression showed prostate restricted expression. Itsstrong expression detected in normal prostate and prostate cancertissues and the low expression detected in other normal tissues indicatethat 108P5H8 is therapeutic and prophylactic target and a diagnostic andprognostic marker for human cancers.

Example 5

[0460] Production of Recombinant 108P5H8 in Prokaryotic Systems

[0461] To express recombinant 108P5H8 in prokaryotic cells, the full orpartial length 108P5H8 cDNA sequences can be cloned into any one of avariety of expression vectors known in the art. One or more of thefollowing regions of 108P5H8 are expressed in these constructs, aminoacids 1 to 429 of variant 1 or variant 2; or amino acids 1 to 388 ofvariant 4, or any 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22,23, 24, 25, 26, 27, 28, 29, 30 or more contiguous amino acids from108P5H8, variants, or analogs thereof. In certain embodiments a regionof 108P5H8 is expressed that encodes an amino acid not shared amongst atleast one of the variants, such as a construct encoding the D to Emutation at amino acid 30.

[0462] A. In Vitro Transcription and Translation Constructs:

[0463] pCRII: To generate 108P5H8 sense and anti-sense RNA probes forRNA in situ investigations, pCRII constructs (Invitrogen, CarlsbadCalif.) are generated encoding either all or fragments of a 108P5H8cDNA. The pCRII vector has Sp6 and T7 promoters flanking the insert todrive the transcription of 108P5H8 RNA for use as probes in RNA in situhybridization experiments. These probes are used to analyze the cell andtissue expression of 108P5H8 at the RNA level. Transcribed 108P5H8 RNArepresenting the cDNA amino acid coding region of the 108P5H8 gene isused in in vitro translation systems such as the TnT™ CoupledReticulolysate System (Promega, Corp., Madison, Wis.) to synthesize108P5H8 protein.

[0464] B. Bacterial Constructs:

[0465] pGEX Constructs: To generate recombinant 108P5H8 proteins inbacteria that are fused to the Glutathione S-transferase (GST) protein,all or parts of a 108P5H8 cDNA protein coding sequence are fused to theGST gene by cloning into pGEX-6P-1 or any other GST-fusion vector of thepGEX family (Amersham Pharmacia Biotech, Piscataway, N.J.). Theseconstructs allow controlled expression of recombinant 108P5H8 proteinsequences with GST fused at the amino-terminus and a six histidineepitope (6× His) at the carboxyl-terminus. The GST and 6× His tagspermit purification of the recombinant fusion protein from inducedbacteria with the appropriate affinity matrix and allow recognition ofthe fusion protein with anti-GST and anti-His antibodies. The 6× His tagis generated by adding 6 histidine codons to the cloning primer at the3′ end, e.g., of the open reading frame (ORF). A proteolytic cleavagesite, such as the PreScission™ recognition site in pGEX-6P-1, may beemployed such that it permits cleavage of the GST tag from108P5H8-related protein. The ampicillin resistance gene and pBR322origin permits selection and maintenance of the pGEX plasmids in E.coli.

[0466] In one embodiment, a GST-fusion protein was constructed andexpressed that encoded amino acids 1-112. This protein was used as animmunogen for generation of a 108P5H8 specific polyclonal antibody asdescribed in example 8.

[0467] pMAL Constructs: To generate, in bacteria, recombinant 108P5H8proteins that are fused to maltose-binding protein (MBP), all or partsof a 108P5H8 cDNA protein coding sequence are fused to the MBP gene bycloning into the pMAL-c2× and pMAL-p2× vectors (New England Biolabs,Beverly, Mass.). These constructs allow controlled expression ofrecombinant 108P5H8 protein sequences with MBP fused at theamino-terminus and a 6× His epitope tag at the carboxyl-terminus. TheMBP and 6× His tags permit purification of the recombinant protein frominduced bacteria with the appropriate affinity matrix and allowrecognition of the fusion protein with anti-MBP and anti-His antibodies.The 6× His epitope tag is generated by adding 6 histidine codons to the3′ cloning primer. A Factor Xa recognition site permits cleavage of thepMAL tag from 108P5H8. The pMAL-c2× and pMAL-p2× vectors are optimizedto express the recombinant protein in the cytoplasm or periplasmrespectively. Periplasm expression enhances folding of proteins withdisulfide bonds.

[0468] pET Constructs: To express 108P5H8 in bacterial cells, all orparts of a 108P5H8 cDNA protein coding sequence are cloned into the pETfamily of vectors (Novagen, Madison, Wis.). These vectors allow tightlycontrolled expression of recombinant 108P5H8 protein in bacteria withand without fusion to proteins that enhance solubility, such as NusA andthioredoxin (Trx), and epitope tags, such as 6× His and S-Tag™ that aidpurification and detection of the recombinant protein. For example,constructs are made utilizing pET NusA fusion system 43.1 such thatregions of a 108P5H8 protein are expressed as amino-terminal fusions toNusA.

[0469] C. Yeast Constructs:

[0470] pESC Constructs: To express 108P5H8 in the yeast speciesSaccharomyces cerevisiae for generation of recombinant protein andfunctional studies, all or parts of a 108P5H8 cDNA protein codingsequence are cloned into the pESC family of vectors each of whichcontain 1 of 4 selectable markers, HIS3, TRP1, LEU2, and URA3(Stratagene, La Jolla, Calif.). These vectors allow controlledexpression from the same plasmid of up to 2 different genes or clonedsequences containing either Flag™ or Myc epitope tags in the same yeastcell. This system is useful to confirm protein-protein interactions of108P5H8. In addition, expression in yeast yields similarpost-translational modifications, such as glycosylations andphosphorylations, that are found when expressed in eukaryotic cells.

[0471] pESP Constructs: To express 108P5H8 in the yeast speciesSaccharomyces pombe, all or parts of a 108P5H8 cDNA protein codingsequence are cloned into the pESP family of vectors. These vectors allowcontrolled high level of expression of a 108P5H8 protein sequence thatis fused at either the amino terminus or at the carboxyl terminus to GSTwhich aids purification of the recombinant protein. A Flag™ epitope tagallows detection of the recombinant protein with anti-Flag™ antibody.

Example 6

[0472] Production of Recombinant 108P5H8 in Eukaryotic Systems

[0473] A. Mammalian Constructs:

[0474] To express recombinant 108P5H8 in eukaryotic cells, the full orpartial length 108P5H8 cDNA sequences can be cloned into any one of avariety of expression vectors known in the art. One or more of thefollowing regions of 108P5H8 are expressed in these constructs, aminoacids 1 to 429 of variant, variant 2 or variant 3; or any 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30 or more contiguous amino acids from 108P5H8, variants, or analogsthereof. In certain embodiments a region of 108P5H8 is expressed thatencodes an amino acid not shared amongst at least two variants.

[0475] The constructs can be transfected into any one of a wide varietyof mammalian cells such as 293T cells. Transfected 293T cell lysates areprobed with an anti-His epitope tag antibody or with anti-108P5H8polyclonal antibodies to verify protein expression.

[0476] pcDNA4/HisMax Constructs: To express 108P5H8 in mammalian cells,a 108P5H8 ORF, or portions thereof, of 108P5H8 are cloned intopcDNA4/HisMax Version A (Invitrogen, Carlsbad, Calif.). Proteinexpression is driven from the cytomegalovirus (CMV) promoter and theSP16 translational enhancer. The recombinant protein has Xpress™ and sixhistidine (6× His) epitopes fused to the amino-terminus. ThepcDNA4/HisMax vector also contains the bovine growth hormone (BGH)polyadenylation signal and transcription termination sequence to enhancemRNA stability along with the SV40 origin for episomal replication andsimple vector rescue in cell lines expressing the large T antigen. TheZeocin resistance gene allows for selection of mammalian cellsexpressing the protein and the ampicillin resistance gene and ColE1origin permits selection and maintenance of the plasmid in E. coli.

[0477] pcDNA3.1/MycHis Constructs: To express 108P5H8 in mammaliancells, a 108P5H8 ORF, or portions thereof, of 108P5H8 with a consensusKozak translation initiation site were cloned into pcDNA3.1/MycHisVersion A (Invitrogen, Carlsbad, Calif.). Protein expression is drivenfrom the cytomegalovirus (CMV) promoter. The recombinant proteins havethe myc epitope and 6× His epitope fused to the carboxyl-terminus. ThepcDNA3.1/MycHis vector also contains the bovine growth hormone (BGH)polyadenylation signal and transcription termination sequence to enhancemRNA stability, along with the SV40 origin for episomal replication andsimple vector rescue in cell lines expressing the large T antigen. TheNeomycin resistance gene can be used, as it allows for selection ofmammalian cells expressing the protein and the ampicillin resistancegene and ColE1 origin permits selection and maintenance of the plasmidin E. coli. The pcDNA3.1/MycHis construct encoding 108P5H8 wastransfected into 293T cells. Expression of 108P5H8 was assayed by flowcytometry and using anti-His antibody as well as polyclonal anti-108P5H8antibody (FIG. 22). Results show that 108P5H8 protein was expressed andwas localized to the cell surface.

[0478] pcDNA3.1/CT-GFP-TOPO Construct: To express 108P5H8 in mammaliancells and to allow detection of the recombinant proteins usingfluorescence, a 108P5H8 ORF, or portions thereof, with a consensus Kozaktranslation initiation site are cloned into pcDNA3.1/CT-GFP-TOPO(Invitrogen, Calif.). Protein expression is driven from thecytomegalovirus (CMV) promoter. The recombinant proteins have the GreenFluorescent Protein (GFP) fused to the carboxyl-terminus facilitatingnon-invasive, in vivo detection and cell biology studies. ThepcDNA3.1CT-GFP-TOPO vector also contains the bovine growth hormone (BGH)polyadenylation signal and transcription termination sequence to enhancemRNA stability along with the SV40 origin for episomal replication andsimple vector rescue in cell lines expressing the large T antigen. TheNeomycin resistance gene allows for selection of mammalian cells thatexpress the protein, and the ampicillin resistance gene and ColE1 originpermits selection and maintenance of the plasmid in E. coli. Additionalconstructs with an amino-terminal GFP fusion are made inpcDNA3.1/NT-GFP-TOPO spanning the entire length of a 108P5H8 protein.

[0479] PAPtag: A 108P5H8 ORF, or portions thereof, is cloned intopAPtag-5 (GenHunter Corp. Nashville, Tenn.). This construct generates analkaline phosphatase fusion at the carboxyl-terminus of a 108P5H8protein while fusing the IgGκ signal sequence to the amino-terminus.Constructs are also generated in which alkaline phosphatase with anamino-terminal IgGκ signal sequence is fused to the amino-terminus of a108P5H8 protein. The resulting recombinant 108P5H8 proteins areoptimized for secretion into the media of transfected mammalian cellsand can be used to identify proteins such as ligands or receptors thatinteract with 108P5H8 proteins. Protein expression is driven from theCMV promoter and the recombinant proteins also contain myc and 6× Hisepitopes fused at the carboxyl-terminus that facilitates detection andpurification. The Zeocin resistance gene present, in the vector allowsfor selection of mammalian cells expressing the recombinant protein andthe ampicillin resistance gene permits selection of the plasmid in E.coli.

[0480] ptag5: A 108P5H8 ORF, or portions thereof, is cloned into pTag-5.This vector is similar to pAPtag but without the alkaline phosphatasefusion. This construct generates 108P5H8 protein with an amino-terminalIgGκ signal sequence and myc and 6× His epitope tags at thecarboxyl-terminus that facilitate detection and affinity purification.The resulting recombinant 108P5H8 protein is optimized for secretioninto the media of transfected mammalian cells, and is used as immunogenor ligand to identify proteins such as ligands or receptors thatinteract with the 108P5H8 proteins. Protein expression is driven fromthe CMV promoter. The Zeocin resistance gene present in the vectorallows for selection of mammalian cells expressing the protein, and theampicillin resistance gene permits selection of the plasmid in E. coli.

[0481] PsecFc: A 108P5H8 ORF, or portions thereof, is also cloned intopsecFc. The psecFc vector was assembled by cloning the humanimmunoglobulin G1 (IgG) Fc (hinge, CH2, CH3 regions) into pSecTag2(Invitrogen, Calif.). This construct generates an IgG1 Fc fusion at thecarboxyl-terminus of the 108P5H8 proteins, while fusing the IgGK signalsequence to N-terminus. 108P5H8 fusions utilizing the murine IgG1 Fcregion are also used. The resulting recombinant 108P5H8 proteins areoptimized for secretion into the media of transfected mammalian cells,and can be used as immunogens or to identify proteins such as ligands orreceptors that interact with 108P5H8 protein. Protein expression isdriven from the CMV promoter. The hygromycin resistance gene present inthe vector allows for selection of mammalian cells that express therecombinant protein, and the ampicillin resistance gene permitsselection of the plasmid in E. coli.

[0482] pSRα Constructs: To generate mammalian cell lines that express108P5H8 constitutively, 108P5H8 ORF, or portions thereof, of 108P5H8were cloned into pSRα constructs. Amphotropic and ecotropic retrovirusesare generated by transfection of pSRα constructs into the 293T-10A1packaging line or co-transfection of pSRα and a helper plasmid(containing deleted packaging sequences) into the 293 cells,respectively. The retrovirus is used to infect a variety of mammaliancell lines, resulting in the integration of the cloned gene, 108P5H8,into the host cell-lines. Protein expression is driven from a longterminal repeat (LTR). The Neomycin resistance gene present in thevector allows for selection of mammalian cells that express the protein,and the ampicillin resistance gene and ColE1 origin permit selection andmaintenance of the plasmid in E. coli. The retroviral vectors canthereafter be used for infection and generation of various cell linesusing, for example, PC3, NIH 3T3, TsuPr1, 293 or rat-1 cells. Results ofexpression of 108P5H8 protein driven from the pSRα in PC3 and NIH3T3cells are shown in FIG. 24.

[0483] Additional pSRα constructs are made that fuse an epitope tag suchas the FLAG™ tag to the carboxyl-terminus of 108P5H8 sequences to allowdetection using anti-Flag antibodies. For example, the FLAG™ sequence 5′gat tac aag gat gac gac gat aag 3′ (SEQ. ID. No.: 2598) is added tocloning primer at the 3′ end of the ORF. Additional pSRα constructs aremade to produce both amino-terminal and carboxyl-terminal GFP and myc/6×His fusion proteins of the full-length 108P5H8 proteins.

[0484] Additional Viral Vectors: Additional constructs are made forviral-mediated delivery and expression of 108P5H8. High virus titerleading to high level expression of 108P5H8 is achieved in viraldelivery systems such as adenoviral vectors and herpes amplicon vectors.A 108P5H8 coding sequences or fragments thereof are amplified by PCR andsubcloned into the AdEasy shuttle vector (Stratagene). Recombination andvirus packaging are performed according to the manufacturer'sinstructions to generate adenoviral vectors. Alternatively, 108P5H8coding sequences or fragments thereof are cloned into the HSV-1 vector(Imgenex) to generate herpes viral vectors. The viral vectors arethereafter used for infection of various cell lines such as PC3, NIH3T3, 293 or rat-1 cells.

[0485] Regulated Expression Systems: To control expression of 108P5H8 inmammalian cells, coding sequences of 108P5H8, or portions thereof, arecloned into regulated mammalian expression systems such as the T-RexSystem (Invitrogen), the GeneSwitch System (Invitrogen) and thetightly-regulated Ecdysone System (Sratagene). These systems allow thestudy of the temporal and concentration dependent effects of recombinant108P5H8. These vectors are thereafter used to control expression of108P5H8 in various cell lines such as PC3, NIH 3T3, 293 or rat-1 cells.

[0486] B. Baculovirus Expression Systems

[0487] To generate recombinant 108P5H8 proteins in a baculovirusexpression system, 108P5H8 ORF, or portions thereof, are cloned into thebaculovirus transfer vector pBlueBac 4.5 (Invitrogen), which provides aHis-tag at the N-terminus. Specifically, pBlueBac-108P5H8 isco-transfected with helper plasmid pBac-N-Blue (Invitrogen) into SF9(Spodoptera frugiperda) insect cells to generate recombinant baculovirus(see Invitrogen instruction manual for details). Baculovirus is thencollected from cell supernatant and purified by plaque assay.

[0488] Recombinant 108P5H8 protein is then generated by infection ofHighFive insect cells (Invitrogen) with purified baculovirus.Recombinant 108P5H8 protein can be detected using anti-108P5H8 oranti-His-tag antibody. 108P5H8 protein can be purified and used invarious cell-based assays or as immunogen to generate polyclonal andmonoclonal antibodies specific for 108P5H8.

Example 7

[0489] Antigenicity Profiles and Secondary Structure

[0490]FIG. 5, FIG. 6, FIG. 7, FIG. 8, and FIG. 9 depict graphically fiveamino acid profiles of the 108P5H8 amino acid sequence (variant 1), eachassessment is available by accessing the ProtScale website (URLwww.expasy.ch/cgi-bin/protscale.p1) on the ExPasy molecular biologyserver.

[0491] These profiles: FIG. 5, Hydrophilicity, (Hopp T. P., Woods K. R.,1981. Proc. Natl. Acad. Sci. U.S.A. 78:3824-3828); FIG. 6,Hydropathicity, (Kyte J., Doolittle R. F., 1982. J. Mol. Biol.157:105-132); FIG. 7, Percentage Accessible Residues (Janin J., 1979Nature 277:491-492); FIG. 8, Average Flexibility, (Bhaskaran R., andPonnuswamy P. K., 1988. Int. J. Pept. Protein Res. 32:242-255); FIG. 9,Beta-turn (Deleage, G., Roux B. 1987 Protein Engineering 1:289-294); andoptionally others available in the art, such as on the ProtScalewebsite, were used to identify antigenic regions of 108P5H8 protein.Each of the above amino acid profiles of 108P5H8 were generated usingthe following ProtScale parameters for analysis: 1) A window size of 9;2) 100% weight of the window edges compared to the window center; and,3) amino acid profile values normalized to lie between 0 and 1.

[0492] Hydrophilicity (FIG. 5), Hydropathicity (FIG. 6) and PercentageAccessible Residues (FIG. 7) profiles were used to determine stretchesof hydrophilic amino acids (i.e., values greater than 0.5 on theHydrophilicity and Percentage Accessible Residues profiles, and valuesless than 0.5 on the Hydropathicity profile). Such regions are likely tobe exposed to the aqueous environment, be present on the surface of theprotein, and thus available for immune recognition, such as byantibodies.

[0493] Average Flexibility (FIG. 8) and Beta-turn (FIG. 9) profilesdetermine stretches of amino acids (i.e., values greater than 0.5 on theBeta-turn profile and the Average Flexibility profile) that are notconstrained in secondary structures such as beta sheets and alphahelices. Such regions are also more likely to be exposed on the proteinand thus accessible to immune recognition, such as by antibodies.

[0494] Antigenic sequences of the full length 108P5H8 protein (variant1) indicated, e.g., by the profiles set forth in FIG. 5, FIG. 6, FIG. 7,FIG. 8, and/or FIG. 9 are used to prepare immunogens, either peptides ornucleic acids that encode them, to generate therapeutic and diagnosticanti-108P5H8 antibodies. The immunogen can be any 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45,50 or more than 50 contiguous amino acids, or the corresponding nucleicacids that encode them, from 108P5H8 protein. In particular, peptideimmunogens of the invention can comprise, a peptide region of at least 5amino acids of FIG. 2 in any whole number increment up to 429 thatincludes an amino acid position having a value greater than 0.5 in theHydrophilicity profile of FIG. 5; a peptide region of at least 5 aminoacids of FIG. 2 in any whole number increment up to 429 that includes anamino acid position having a value less than 0.5 in the Hydropathicityprofile of FIG. 6; a peptide region of at least 5 amino acids of FIG. 2in any whole number increment up to 429 that includes an amino acidposition having a value greater than 0.5 in the Percent AccessibleResidues profile of FIG. 7; a peptide region of at least 5 amino acidsof FIG. 2 in any whole number increment up to 429 that includes an aminoacid position having a value greater than 0.5 in the Average Flexibilityprofile on FIG. 8; and, a peptide region of at least 5 amino acids ofFIG. 2 in any whole number increment up to 429 that includes an aminoacid position having a value greater than 0.5 in the Beta-turn profileof FIG. 9. Peptide immunogens of the invention can also comprise nucleicacids that encode any of the forgoing.

[0495] All immunogens of the invention, peptide or nucleic acid, can beembodied in human unit dose form, or comprised by a composition thatincludes a pharmaceutical excipient compatible with human physiology.

[0496] The secondary structure of 108P5H8, namely the predicted presenceand location of alpha helices, extended strands, and random coils, ispredicted from the primary amino acid sequence of 108P5H8 variant 1using the HNN—Hierarchical Neural Network method (Guermeur, 1997,http://pbil.ibcp.fr/cgi-bin/npsa_automat.p1?page=npsa_nn.html), accessedfrom the ExPasy molecular biology server (http://www.expasy.ch/tools/).The analysis indicates that 108P5H8 is composed of 49.88% alpha helix,11.66% extended strand, and 38.46% random coil (FIG. 18).

[0497] Analysis for the potential presence of transmembrane domains in108P5H8 was carried out using a variety of transmembrane predictionalgorithms accessed from the ExPasy molecular biology server((http://www.expasy.ch/tools/). The programs predict the presence of 6transmembrane domains in 108P5H8. Shown graphically in FIG. 19A and 19Bare the results of analysis using the TMpred and TMHMM predictionprograms, respectively, depicting the location of the 6 transmembranedomains. The results of each program, namely the amino acids encodingthe transmembrane domains are summarized in Table XXI.

Example 8

[0498] Generation of 108P5H8 Polyclonal Antibodies

[0499] Polyclonal antibodies can be raised in a mammal, for example, byone or more injections of an immunizing agent and, if desired, anadjuvant. Typically, the immunizing agent and/or adjuvant will beinjected in the mammal by multiple subcutaneous or intraperitonealinjections. In addition to immunizing with the full length 108P5H8protein, computer algorithms are employed in design of immunogens that,based on amino acid sequence analysis contain characteristics of beingantigenic and available for recognition by the immune system of theimmunized host (see the Example entitled “Antigenicity Profiles andSecondary Structure”). Such regions would be predicted to behydrophilic, flexible, in beta-turn conformations, and be exposed on thesurface of the protein (see, e.g., FIG. 5, FIG. 6, FIG. 7, FIG. 8, orFIG. 9 for amino acid profiles that indicate such regions of 108P5H8).

[0500] For example, 108P5H8 recombinant bacterial fusion proteins orpeptides containing hydrophilic, flexible, beta-turn regions of 108P5H8are used as antigens to generate polyclonal antibodies in New ZealandWhite rabbits. Such regions often reside in extracellular andintracellular loops between transmembrane domains. For example, suchregions include, but are not limited to, amino acids 1-112(intracellular amino terminus), amino acids 139-152 (1^(st)extracellular loop), amino acids 201-214 (second extracellular loop),amino acids 294-307 (third extracellular loop), or amino acids 336-429(carboxyl terminus). It is useful to conjugate the immunizing agent to aprotein known to be immunogenic in the mammal being immunized. Examplesof such immunogenic proteins include, but are not limited to, keyholelimpet hemocyanin (KLH), serum albumin, bovine thyroglobulin, andsoybean trypsin inhibitor. In one embodiment, a peptide encoding aminoacids 294-307 of 108P5H8 is conjugated to KLH and used to immunize therabbit. Alternatively the immunizing agent may include all or portionsof a 108P5H8 protein, analogs or fusion proteins thereof. For example, a108P5H8 amino acid sequence can be fused using recombinant DNAtechniques to any one of a variety of fusion protein partners that arewell known in the art, such as glutathione-S-transferase (GST) and HIStagged fusion proteins. Such fusion proteins are purified from inducedbacteria using the appropriate affinity matrix.

[0501] In one embodiment, a GST-fusion protein encoding amino acids1-112 of 108P5H8 was produced and purified and used to immunize arabbit. This polyclonal antibody specifically recognized bothrecombinant and endogenous 108P5H8 protein in cells and tissues. FIG. 20shows non-androgen-regulated expression of 108P5H8 in the prostatecancer cell lines LNCaP and LAPC4. The expression of 108P5H8 is cellsurface as detected by the polyclonal antibody in LNCaP and LAPC4 cells(FIG. 21) and when overexpressed in 293T cells (FIG. 22). FIG. 23 showsexpression in prostate patient cancer samples, including metastaticdisease, indicating the protein is a therapeutic target in bothandrogen-dependent and independent prostate cancer. 108P5H8 is alsoexpressed in ovarian cancer, but not in normal ovary (FIG. 23),indicating the protein is a therapeutic and diagnostic target in thisdisease as well.

[0502] In addition to GST-fusions, other recombinant bacterial fusionproteins that can be employed include maltose binding protein, LacZ,thioredoxin, NusA, or an immunoglobulin constant region (see the sectionentitled “Production of 108P5H8 in Prokaryotic Systems” and CurrentProtocols In Molecular Biology, Volume 2, Unit 16, Frederick M. Ausubulet al. eds., 1995; Linsley, P. S., Brady, W., Urnes, M., Grosmaire, L.,Damle, N., and Ledbetter, L. (1991) J. Exp. Med. 174, 561-566).

[0503] In addition to bacterial-derived fusion proteins,mammalian-expressed protein antigens are also used. These antigens areexpressed from mammalian expression vectors such as the Tag5 andFc-fusion vectors (see the section entitled “Production of Recombinant108P5H8 in Eukaryotic Systems”), and retain post-translationalmodifications such as glycosylations found in native protein. In oneembodiment, the first extracellular loop of 108P5H8 (amino acids139-152) is cloned into the Tag5 mammalian secretion-vector. Therecombinant protein is purified by metal chelate chromatography fromtissue culture supernatants of 293T cells stably expressing therecombinant vector. The purified Tag5 108P5H8 protein is then used asimmunogen.

[0504] During the immunization protocol, it is useful to mix or emulsifythe antigen in adjuvants that enhance the immune response of the hostanimal. Examples of adjuvants include, but are not limited to, completeFreund's adjuvant (CFA) and MPL-TDM adjuvant (monophosphoryl Lipid A,synthetic trehalose dicorynomycolate).

[0505] In a typical protocol, rabbits are initially immunizedsubcutaneously with up to 200 μg, typically 100-200 μg, of fusionprotein or peptide conjugated to KLH mixed in complete Freund's adjuvant(CFA). Rabbits are then injected subcutaneously every two weeks with upto 200 μg, typically 100-200 μg, of the immunogen in incomplete Freund'sadjuvant (IFA). Test bleeds are taken approximately 7-10 days followingeach immunization and used to monitor the titer of the antiserum byELISA.

[0506] To test reactivity and specificity of immune serum, such as therabbit serum derived from immunization with Tag5 108P5H8 protein orKLH-coupled peptide encoding amino acids 294-307, the full-length108P5H8 cDNA is cloned into pcDNA 3.1 myc-his expression vector(Invitrogen, see the Example entitled “Production of Recombinant 108P5H8in Eukaryotic Systems”). After transfection of the constructs into 293Tcells, cell lysates are probed with the anti-108P5H8 serum and withanti-His antibody (Santa Cruz Biotechnologies, Santa Cruz, Calif.) todetermine specific reactivity to denatured 108P5H8 protein using theWestern blot technique (FIG. 20). Immunoprecipitation and flowcytometric analyses of 293T and other recombinant 108P5H8-expressingcells determine recognition of native protein by the antiserum (FIG. 22,FIG. 24). In addition, Western blot, immunoprecipitation, fluorescentmicroscopy, and flow cytometric techniques using cells that endogenouslyexpress 108P5H8 are carried out to test specificity (FIG. 20, FIG. 21).

[0507] The anti-serum from the Tag5 108P5H8 immunized rabbit is affinitypurified by passage over a column composed of the Tag5 antigencovalently coupled to Affigel matrix (BioRad, Hercules, Calif.). Theserum is then further purified by protein G affinity chromatography toisolate the IgG fraction. Serum from rabbits immunized with theGST-fusion protein was purified by depletion of antibodies reactive tothe fusion partner sequence (GST) by passage over an affinity columncontaining the GST alone and then by passage back over a GST-108P5H8column to isolate gene specific antibodies. Sera from other His-taggedantigens and peptide immunized rabbits as well as fusion partnerdepleted sera are affinity purified by passage over a column matrixcomposed of the original protein immunogen or free peptide.

Example 9

[0508] Generation of 108P5H8 Monoclonal Antibodies (mAbs)

[0509] In one embodiment, therapeutic mAbs to 108P5H8 comprise thosethat react with epitopes of the protein that would disrupt or modulatethe biological function of 108P5H8, for example those that would disruptits interaction with ligands, proteins, or substrates that mediate itsbiological activity. Immunogens for generation of such mAbs includethose designed to encode or contain an entire 108P5H8 protein or itsvariants or regions of 108P5H8 protein predicted to be exposed on thecell surface and/or antigenic from computer analysis of the amino acidsequence (see, e.g., FIG. 5, FIG. 6, FIG. 7, FIG. 8, or FIG. 9, and theExample entitled “Antigenicity Profiles and Secondary Structure”).Immunogens include peptides, recombinant bacterial proteins, andmammalian expressed Tag 5 proteins and human and murine IgG FC fusionproteins. In addition, cells expressing high levels of 108P5H8, such as293T-108P5H8 or 300.19-108P5H8 murine Pre-B cells, are used to immunizemice.

[0510] To generate mAbs to 108P5H8, mice are first immunizedintraperitoneally (IP) with, typically, 10-50 μg of protein immunogen or10⁷ 108P5H8-expressing cells mixed in complete Freund's adjuvant. Miceare then subsequently immunized IP every 2-4 weeks with, typically,10-50 μg of protein immunogen or 10⁷ cells mixed in incomplete Freund'sadjuvant. Alternatively, MPL-TDM adjuvant is used in immunizations. Inaddition to the above protein and cell-based immunization strategies, aDNA-based immunization protocol is employed in which a mammalianexpression vector encoding 108P5H8 sequence is used to immunize mice bydirect injection of the plasmid DNA. For example, the predicted thirdextracellular loop, amino acids 294-307 of 108P5H8, is cloned into theTag5 mammalian secretion vector and the recombinant vector is used asimmunogen. In another example the amino acids are cloned into anFc-fusion secretion vector in which a 108P5H8 sequence is fused at theamino-terminus to an IgK leader sequence and at the carboxyl-terminus tothe coding sequence of the human or murine IgG Fc region. Thisrecombinant vector is then used as immunogen. The plasmid immunizationprotocols are used in combination with purified proteins expressed fromthe same vector and with cells expressing 108P5H8.

[0511] During the immunization protocol, test bleeds are taken 7-10 daysfollowing an injection to monitor titer and specificity of the immuneresponse. Once appropriate reactivity and specificity is obtained asdetermined by ELISA, Western blotting, immunoprecipitation, fluorescencemicroscopy, and flow cytometric analyses, fusion and hybridomageneration is then carried out with established procedures well known inthe art (see, e.g., Harlow and Lane, 1988).

[0512] In one embodiment, monoclonal antibodies are derived fromimmunization of mice with 300.19 cells engineered to express high levelsof 108P5H8 (>30,000 molecules per cell). Balb C mice are initiallyimmunized intraperitoneally with 10⁷ cells mixed in complete Freund'sadjuvant. Mice are subsequently immunized every two weeks with 10⁷ cellsmixed in incomplete Freund's adjuvant for a total of threeimmunizations. Reactivity and specificity of serum to the full length108P5H8 protein is monitored by Western blotting, immunoprecipitationand flow cytometry using various cells engineered to overexpress 108P5H8protein (FIG. 24). Mice showing the strongest reactivity are rested andgiven a final injection of cells in PBS and then sacrificed four dayslater. The spleens of the sacrificed mice are harvested and fused toSPO/2 myeloma cells using standard procedures (see, e.g., Harlow andLane, 1988). Supernatants from HAT selected growth wells are, screenedby ELISA, Western blot, immunoprecipitation, fluorescent microscopy, andflow cytometry to identify 108P5H8 specific antibody-producing clones.

[0513] The binding affinity of a 108P5H8 monoclonal antibody isdetermined using standard technologies. Affinity measurements quantifythe strength of antibody to epitope binding and are used to help definewhich 108P5H8 monoclonal antibodies preferred, e.g., for diagnostic ortherapeutic use, as appreciated by one of skill in the art. The BIAcoresystem (Uppsala, Sweden) is a preferred method for determining bindingaffinity. The BIAcore system uses surface plasmon resonance (SPR,Welford K. 1991, Opt. Quant. Elect. 23: 1; Morton and Myszka, 1998,Methods in Enzymology 295: 268) to monitor biomolecular interactions inreal time. BIAcore analysis conveniently generates association rateconstants, dissociation rate constants, equilibrium dissociationconstants, and affinity constants.

Example 10

[0514] HLA Class I and Class II Binding Assays

[0515] HLA class I and class II binding assays using purified HLAmolecules are performed in accordance with disclosed protocols (e.g.,PCT publications WO 94/20127 and WO 94/03205; Sidney et al., CurrentProtocols in Immunology 18.3.1 (1998); Sidney, et al., J. Immunol.154:247 (1995); Sette, et al., Mol. Immunol. 31:813 (1994)). Briefly,purified MHC molecules (5 to 500 nM) are incubated with variousunlabeled peptide inhibitors and 1-10 nM ¹²⁵I-radiolabeled probepeptides as described. Following incubation, MHC-peptide complexes areseparated from free peptide by gel filtration and the fraction ofpeptide bound is determined. Typically, in preliminary experiments, eachMHC preparation is titered in the presence of fixed amounts ofradiolabeled peptides to determine the concentration of HLA moleculesnecessary to bind 10-20% of the total radioactivity. All subsequentinhibition and direct binding assays are performed using these HLAconcentrations.

[0516] Since under these conditions [label]<[HLA] and IC₅₀≧[HLA], themeasured IC₅₀ values are reasonable approximations of the true K_(D)values. Peptide inhibitors are typically tested at concentrationsranging from 120 μg/ml to 1.2 ng/ml, and are tested in two to fourcompletely independent experiments. To allow comparison of the dataobtained in different experiments, a relative binding figure iscalculated for each peptide by dividing the IC₅₀ of a positive controlfor inhibition by the IC₅₀ for each tested peptide (typically unlabeledversions of the radiolabeled probe peptide). For database purposes, andinter-experiment comparisons, relative binding values are compiled.These values can subsequently be converted back into IC50 nM values bydividing the IC₅₀ nM of the positive controls for inhibition by therelative binding of the peptide of interest. This method of datacompilation is accurate and consistent for comparing peptides that havebeen tested on different days, or with different lots of purified MHC.

[0517] Binding assays as outlined above may be used to analyze HLAsupermotif and/or HLA motif-bearing peptides.

Example 11

[0518] Identification of HLA Supermotif- and Motif-Bearing CTL CandidateEpitopes

[0519] HLA vaccine compositions of the invention can include multipleepitopes. The multiple epitopes can comprise multiple HLA supermotifs ormotifs to achieve broad population coverage. This example illustratesthe identification and confirmation of supermotif- and motif-bearingepitopes for the inclusion in such a vaccine composition. Calculation ofpopulation coverage is performed using the strategy described below.

[0520] Computer Searches and Algorithms for Identification of Supermotifand/or Motif-Bearing Epitopes

[0521] The searches performed to identify the motif-bearing peptidesequences in the Example entitled “Antigenicity Profiles” and TablesV-XVIII, XXII, and XXIII employ the protein sequence data from the geneproduct of 108P5H8 set forth in FIGS. 2 and 3.

[0522] Computer searches for epitopes bearing HLA Class I or Class IIsupermotifs or motifs are performed as follows. All translated 108P5H8protein sequences are analyzed using a text string search softwareprogram to identify potential peptide sequences containing appropriateHLA binding motifs; such programs are readily produced in accordancewith information in the art in view of known motif/supermotifdisclosures. Furthermore, such calculations can be made mentally.

[0523] Identified A2-, A3-, and DR-supermotif sequences are scored usingpolynomial algorithms to predict their capacity to bind to specificHLA-Class I or Class II molecules. These polynomial algorithms accountfor the impact of different amino acids at different positions, and areessentially based on the premise that the overall affinity (or ΔG) ofpeptide-HLA molecule interactions can be approximated as a linearpolynomial function of the type:

“ΔG”=a _(1i) ×a _(2i) ×a _(3i) . . . ×a _(ni)

[0524] where a_(ji) is a coefficient which represents the effect of thepresence of a given amino acid (j) at a given position (i) along thesequence of a peptide of n amino acids. The crucial assumption of thismethod is that the effects at each position are essentially independentof each other (i.e., independent binding of individual side-chains).When residue j occurs at position i in the peptide, it is assumed tocontribute a constant amount j_(i) to the free energy of binding of thepeptide irrespective of the sequence of the rest of the peptide.

[0525] The method of derivation of specific algorithm coefficients hasbeen described in Gulukota et al., J. Mol. Biol 267:1258-126, 1997; (seealso Sidney et al., Human Immunol. 45:79-93, 1996; and Southwood et al.,J. Immunol. 160:3363-3373, 1998). Briefly, for all i positions, anchorand non-anchor alike, the geometric mean of the average relative binding(ARB) of all peptides carrying j is calculated relative to the remainderof the group, and used as the estimate of j_(i). For Class II peptides,if multiple alignments are possible, only the highest scoring alignmentis utilized, following an iterative procedure. To calculate an algorithmscore of a given peptide in a test set, the ARB values corresponding tothe sequence of the peptide are multiplied. If this product exceeds achosen threshold, the peptide is predicted to bind. Appropriatethresholds are chosen as a function of the degree of stringency ofprediction desired.

[0526] Selection of HLA-A2 Supertype Cross-Reactive Peptides

[0527] Protein sequences from 108P5H8 are scanned utilizing motifidentification software, to identify 8-, 9- 10- and 11 -mer sequencescontaining the HLA-A2-supermotif main anchor specificity. Typically,these sequences are then scored using the protocol described above andthe peptides corresponding to the positive-scoring sequences aresynthesized and tested for their capacity to bind purified HLA-A*0201molecules in vitro (HLA-A*0201 is considered a prototype A2 supertypemolecule).

[0528] These peptides are then tested for the capacity to bind toadditional A2-supertype molecules (A*0202, A*0203, A*0206, and A*6802).Peptides that bind to at least three of the five A2-supertype allelestested are typically deemed A2-supertype cross-reactive binders.Preferred peptides bind at an affinity equal to or less than 500 nM tothree or more HLA-A2 supertype molecules.

[0529] Selection of HLA-A3 Supermotif-Bearing Epitopes

[0530] The 108P5H8 protein sequence(s) scanned above is also examinedfor the presence of peptides with the HLA-A3-supermotif primary anchors.Peptides corresponding to the HLA A3 supermotif-bearing sequences arethen synthesized and tested for binding to HLA-A*0301 and HLA-A*1101molecules, the molecules encoded by the two most prevalent A3-supertypealleles. The peptides that bind at least one of the two alleles withbinding affinities of ≦500 nM, often ≦200 nM, are then tested forbinding cross-reactivity to the other common A3-supertype alleles (e.g.,A*3101, A*3301, and A*6801) to identify those that can bind at leastthree of the five HLA-A3-supertype molecules tested.

[0531] Selection of HLA-B7 Supermotif Bearing Epitopes

[0532] The 108P5H8 protein(s) scanned above is also analyzed for thepresence of 8-, 9- 10-, or 11-mer peptides with the HLA-B7-supermotif.Corresponding peptides are synthesized and tested for binding toHLA-B*0702, the molecule encoded by the most common B7-supertype allele(i.e., the prototype B7 supertype allele). Peptides binding B*0702 withIC₅₀ of ≦500 nM are identified using standard methods. These peptidesare then tested for binding to other common B7-supertype molecules(e.g., B*3501, B*5101, B*5301, and B*5401). Peptides capable of bindingto three or more of the five B7-supertype alleles tested are therebyidentified.

[0533] Selection of A1 and A24 Motif-Baring Epitopes

[0534] To further increase population coverage, HLA-A1 and -A24 epitopescan also be incorporated into vaccine compositions. An analysis of the108P5H8 protein can also be performed to identify HLA-A1- andA24-motif-containing sequences.

[0535] High affinity and/or cross-reactive binding epitopes that bearother motif and/or supermotifs are identified using analogousmethodology.

Example 12

[0536] Confirmation of Immunogenicity

[0537] Cross-reactive candidate CTL A2-supermotif-bearing peptides thatare identified as described herein are selected to confirm in vitroimmunogenicity. Confirmation is performed using the followingmethodology:

[0538] Target Cell Lines for Cellular Screening:

[0539] The 0.221 A2.1 cell line, produced by transferring the HLA-A2.1gene into the HLA-A, -B, -C null mutant human B-lymphoblastoid cell line721.221, is used as the peptide-loaded target to measure activity ofHLA-A2.1-restricted CTL. This cell line is grown in RPMI-1640 mediumsupplemented with antibiotics, sodium pyruvate, nonessential amino acidsand 10% (v/v) heat inactivated FCS. Cells that express an antigen ofinterest, or transfectants comprising the gene encoding the antigen ofinterest, can be used as target cells to confirm the ability ofpeptide-specific CTLs to recognize endogenous antigen.

[0540] Primary CTL Induction Cultures:

[0541] Generation of Dendritic Cells (DC): PBMCs are thawed in RPMI with30 μg/ml DNAse, washed twice and resuspended in complete medium(RPMI-1640 plus 5% AB human serum, non-essential amino acids, sodiumpyruvate, L-glutamine and penicillin/streptomycin). The monocytes arepurified by plating 10×10⁶ PBMC/well in a 6-well plate. After 2 hours at37° C., the non-adherent cells are removed by gently shaking the platesand aspirating the supernatants. The wells are washed a total of threetimes with 3 ml RPMI to remove most of the non-adherent and looselyadherent cells. Three ml of complete medium containing 50 ng/ml ofGM-CSF and 1,000 U/ml of IL-4 are then added to each well. TNFα is addedto the DCs on day 6 at 75 ng/ml and the cells are used for CTL inductioncultures on day 7.

[0542] Induction of CTL with DC and Peptide: CD8+ T-cells are isolatedby positive selection with Dyna1 immunomagnetic beads (Dynabeads® M-450)and the detacha-bead® reagent. Typically about 200-250×10⁶ PBMC areprocessed to obtain 24×10⁶ CD8⁺ T-cells (enough for a 48-well plateculture). Briefly, the PBMCs are thawed in RPMI with 30 μg/ml DNAse,washed once with PBS containing 1% human AB serum and resuspended inPBS/1% AB serum at a concentration of 20×10⁶ cells/ml. The magneticbeads are washed 3 times with PBS/AB serum, added to the cells (140 μlbeads/20×10⁶ cells) and incubated for 1 hour at 4° C. with continuousmixing. The beads and cells are washed 4× with PBS/AB serum to removethe nonadherent cells and resuspended at 100×10⁶ cells/ml (based on theoriginal cell number) in PBS/AB serum containing 100 μl/ml detacha-bead®reagent and 30 μg/ml DNAse. The mixture is incubated for 1 hour at roomtemperature with continuous mixing. The beads are washed again withPBS/AB/DNAse to collect the CD8+ T-cells. The DC are collected andcentrifuged at 1300 rpm for 5-7 minutes, washed once with PBS with 1%BSA, counted and pulsed with 40 μg/ml of peptide at a cell concentrationof 1-2×10⁶/ml in the presence of 3 μg/ml β₂-microglobulin for 4 hours at20° C. The DC are then irradiated (4,200 rads), washed 1 time withmedium and counted again.

[0543] Setting up induction cultures: 0.25 ml cytokine-generated DC (at1×10⁵ cells/ml) are co-cultured with 0.25 ml of CD8+ T-cells (at 2×10⁶cell/ml) in each well of a 48-well plate in the presence of 10 ng/ml ofIL-7. Recombinant human IL-1 is added the next day at a finalconcentration of 10 ng/ml and rhuman IL-2 is added 48 hours later at 10IU/ml.

[0544] Restimulation of the induction cultures with peptide-pulsedadherent cells: Seven and fourteen days after the primary induction, thecells are restimulated with peptide-pulsed adherent cells. The PBMCs arethawed and washed twice with RPMI and DNAse. The cells are resuspendedat 5×10⁶ cells/ml and irradiated at ˜4200 rads. The PBMCs are plated at2×10⁶ in 0.5 ml complete medium per well and incubated for 2 hours at37° C. The plates are washed twice with RPMI by tapping the plate gentlyto remove the nonadherent cells and the adherent cells pulsed with 10μg/ml of peptide in the presence of 3 μg/ml β₂ microglobulin in 0.25 mlRPMI/5% AB per well for 2 hours at 37° C. Peptide solution from eachwell is aspirated and the wells are washed once with RPMI. Most of themedia is aspirated from the induction cultures (CD8+ cells) and broughtto 0.5 ml with fresh media. The cells are then transferred to the wellscontaining the peptide-pulsed adherent cells. Twenty four hours laterrecombinant human IL-10 is added at a final concentration of 10 ng/mland recombinant human IL2 is added the next day and again 2-3 days laterat 50 IU/ml (Tsai et al., Critical Reviews in Immunology 18(1-2):65-75,1998). Seven days later, the cultures are assayed for CTL activity in a⁵¹Cr release assay. In some experiments the cultures are assayed forpeptide-specific recognition in the in situ IFNγ ELISA at the time ofthe second restimulation followed by assay of endogenous recognition 7days later. After expansion, activity is measured in both assays for aside-by-side comparison.

[0545] Measurement of CTL Lytic Activity by ⁵¹Cr Release.

[0546] Seven days after the second restimulation, cytotoxicity isdetermined in a standard (5 hr) ⁵¹Cr release assay by assayingindividual wells at a single E:T. Peptide-pulsed targets are prepared byincubating the cells with 10 μg/ml peptide overnight at 37° C.

[0547] Adherent target cells are removed from culture flasks withtrypsin-EDTA. Target cells are labeled with 200 μCi of ⁵¹Cr sodiumchromate (Dupont, Wilmington, Del.) for 1 hour at 37° C. Labeled targetcells are resuspended at 10⁶ per ml and diluted 1:10 with K562 cells ata-concentration of 3.3×10⁶/ml (an NK-sensitive erythroblastoma cell lineused to reduce non-specific lysis). Target cells (100 μl) and effectors(100 μl) are plated in 96 well round-bottom plates and incubated for 5hours at 37° C. At that time, 100 μl of supernatant are collected fromeach well and percent lysis is determined according to the formula:

[(cpm of the test sample−cpm of the spontaneous ⁵¹Cr releasesample)/(cpm of the maximal ⁵¹Cr release sample−cpm of the spontaneous⁵¹Cr release sample)]×100.

[0548] Maximum and spontaneous release are determined by incubating thelabeled targets with 1% Triton X-100 and media alone, respectively. Apositive culture is defined as one in which the specific lysis(sample-background) is 10% or higher in the case of individual wells andis 15% or more at the two highest E:T ratios when expanded cultures areassayed.

[0549] In situ Measurement of Human IFNγ Production as an Indicator ofPeptide-Specific and Endogenous Recognition

[0550] Immulon 2 plates are coated with mouse anti-human IFNγ monoclonalantibody (4 μg/ml 0.1M NaHCO₃, pH8.2) overnight at 4° C. The plates arewashed with Ca²⁺, Mg²⁺-free PBS/0.05% Tween 20 and blocked with PBS/10%FCS for two hours, after which the CTLs (100 μl/well) and targets (100μl/well) are added to each well, leaving empty wells for the standardsand blanks (which received media only). The target cells, eitherpeptide-pulsed or endogenous targets, are used at a concentration of1×10⁶ cells/ml. The plates are incubated for 48 hours at 37° C. with 5%CO₂.

[0551] Recombinant human IFN-gamma is added to the standard wellsstarting at 400 pg or 1200 pg/100 microliter/well and the plateincubated for two hours at 37° C. The plates are washed and 100 μl ofbiotinylated mouse anti-human IFN-gamma monoclonal antibody (2microgram/ml in PBS/3% FCS/0.05% Tween 20) are added and incubated for 2hours at room temperature. After washing again, 100 microliterHRP-streptavidin (1:4000) are added and the plates incubated for onehour at room temperature. The plates are then washed 6× with washbuffer, 100 microliter/well developing solution (TMB 1:1) are added, andthe plates allowed to develop for 5-15 minutes. The reaction is stoppedwith 50 microliter/well 1M H₃PO₄ and read at OD450. A culture isconsidered positive if it measured at least 50 pg of IFN-gamma/wellabove background and is twice the background level of expression.

[0552] CTL Expansion.

[0553] Those cultures that demonstrate specific lytic activity againstpeptide-pulsed targets and/or tumor targets are expanded over a two weekperiod with anti-CD3. Briefly, 5×10⁴ CD8+ cells are added to a T25 flaskcontaining the following: 1×10⁶ irradiated (4,200 rad) PBMC (autologousor allogeneic) per ml, 2×10⁵ irradiated (8,000 rad) EBV-transformedcells per ml, and OKT3 (anti-CD3) at 30 ng per ml in RPMI-1640containing 10% (v/v) human AB serum, non-essential amino acids, sodiumpyruvate, 25 μM 2-mercaptoethanol, L-glutamine andpenicillin/streptomycin. Recombinant human IL2 is added 24 hours laterat a final concentration of 200 IU/ml and every three days thereafterwith fresh media at 50 IU/ml. The cells are split if the cellconcentration exceeds 1×10⁶/ml and the cultures are assayed between days13 and 15 at E:T ratios of 30, 10, 3 and 1:1 in the ⁵¹Cr release assayor at 1×10⁶/ml in the in situ IFNγ assay using the same targets asbefore the expansion.

[0554] Cultures are expanded in the absence of anti-CD3⁺ as follows.Those cultures that demonstrate specific lytic activity against peptideand endogenous targets are selected and 5×10⁴ CD8⁺ cells are added to aT25 flask containing the following: 1×10⁶ autologous PBMC per ml whichhave been peptide-pulsed with 10 μg/ml peptide for two hours at 37° C.and irradiated (4,200 rad); 2×10⁵ irradiated (8,000 rad) EBV-transformedcells per ml RPMI-1640 containing 10% (v/v) human AB serum,non-essential AA, sodium pyruvate, 25 mM 2-ME, L-glutamine andgentamicin.

[0555] Immunogenicity of A2 Supermotif-Bearing Peptides

[0556] A2-supermotif cross-reactive binding peptides are tested in thecellular assay for the ability to induce peptide-specific CTL in normalindividuals. In this analysis, a peptide is typically considered to bean epitope if it induces peptide-specific CTLs in at least individuals,and preferably, also recognizes the endogenously expressed peptide.

[0557] Immunogenicity can also be confirmed using PBMCs isolated frompatients bearing a tumor that expresses 108P5H8. Briefly, PBMCs areisolated from patients, re-stimulated with peptide-pulsed monocytes andassayed for the ability to recognize peptide-pulsed target cells as wellas transfected cells endogenously expressing the antigen.

[0558] Evaluation of A*03/A11 Immunogenicity

[0559] HLA-A3 supermotif-bearing cross-reactive binding peptides arealso evaluated for immunogenicity using methodology analogous for thatused to evaluate the immunogenicity of the HLA-A2 supermotif peptides.

[0560] Evaluation of B7 Immunogenicity

[0561] Immunogenicity screening of the B7-supertype cross-reactivebinding peptides identified as set forth herein are confirmed in amanner analogous to the confirmation of A2-and A3-supermotif-bearingpeptides.

[0562] Peptides bearing other supermotifs/motifs, e.g., HLA-A1, HLA-A24etc. are also confirmed using similar methodology

Example 13

[0563] Implementation of the Extended Supermotif to Improve the BindingCapacity of Native Epitopes by Creating Analogs

[0564] HLA motifs and supermotifs (comprising primary and/or secondaryresidues) are useful in the identification and preparation of highlycross-reactive native peptides, as demonstrated herein. Moreover, thedefinition of HLA motifs and supermotifs also allows one to engineerhighly cross-reactive epitopes by identifying residues within a nativepeptide sequence which can be analoged to confer upon the peptidecertain characteristics, e.g. greater cross-reactivity within the groupof HLA molecules that comprise a supertype, and/or greater bindingaffinity for some or all of those HLA molecules. Examples of analogingpeptides to exhibit modulated binding affinity are set forth in thisexample.

[0565] Analoging at Primary Anchor Residues

[0566] Peptide engineering strategies are implemented to furtherincrease the cross-reactivity of the epitopes. For example, the mainanchors of A2-supermotif-bearing peptides are altered, for example, tointroduce a preferred L, I, V, or M at position 2, and I or V at theC-terminus.

[0567] To analyze the cross-reactivity of the analog peptides, eachengineered analog is initially tested for binding to the prototype A2supertype allele A*0201, then, if A*0201 binding capacity is maintained,for A2-supertype cross-reactivity.

[0568] Alternatively, a peptide is confirmed as binding one or allsupertype members and then analoged to modulate binding affinity to anyone (or more) of the supertype members to add population coverage.

[0569] The selection of analogs for immunogenicity in a cellularscreening analysis is typically further restricted by the capacity ofthe parent wild type (WT) peptide to bind at least weakly, i.e., bind atan IC₅₀ of 5000 nM or less, to three of more A2 supertype alleles. Therationale for this requirement is that the WT peptides must be presentendogenously in sufficient quantity to be biologically relevant.Analoged peptides have been shown to have increased immunogenicity andcross-reactivity by T cells specific for the parent epitope (see, e.g.,Parkhurst et al., J. Immunol. 157:2539, 1996; and Pogue et al., Proc.Natl. Acad. Sci. USA 92:8166, 1995).

[0570] In the cellular screening of these peptide analogs, it isimportant to confirm that analog-specific CTLs are also able torecognize the wild-type peptide and, when possible, target cells thatendogenously express the epitope.

[0571] Analoging of HLA-A3 and B7-Supermotif-Bearing Peptides

[0572] Analogs of HLA-A3 supermotif-bearing epitopes are generated usingstrategies similar to those employed in analoging HLA-A2supermotif-bearing peptides. For example, peptides binding to ⅗ of theA3-supertype molecules are engineered at primary anchor residues topossess a preferred residue (V, S, M, or A) at position 2.

[0573] The analog peptides are then tested for the ability to bind A*03and A*11 (prototype A3 supertype alleles). Those peptides thatdemonstrate ≦500 nM binding capacity are then confirmed as havingA3-supertype cross-reactivity.

[0574] Similarly to the A2- and A3-motif bearing peptides, peptidesbinding 3 or more B7-supertype alleles can be improved, where possible,to achieve increased cross-reactive binding or greater binding affinityor binding half life. B7 supermotif-bearing peptides are, for example,engineered to possess a preferred residue (V, I, L, or F) at theC-terminal primary anchor position, as demonstrated by Sidney et al. (J.Immunol. 157:3480-3490, 1996).

[0575] Analoging at primary anchor residues of other motif and/orsupermotif-bearing epitopes is performed in a like manner.

[0576] The analog peptides are then be confirmed for immunogenicity,typically in a cellular screening assay. Again, it is generallyimportant to demonstrate that analog-specific CTLs are also able torecognize the wild-type peptide and, when possible, targets thatendogenously express the epitope.

[0577] Analoging at Secondary Anchor Residues

[0578] Moreover, HLA supermotifs are of value in engineering highlycross-reactive peptides and/or peptides that bind HLA molecules withincreased affinity by identifying particular residues at secondaryanchor positions that are associated with such properties. For example,the binding capacity of a B7 supermotif-bearing peptide with an Fresidue at position 1 is analyzed. The peptide is then analoged to, forexample, substitute L for F at position 1. The analoged peptide isevaluated for increased binding affinity, binding half life and/orincreased cross-reactivity. Such a procedure identifies analogedpeptides with enhanced properties.

[0579] Engineered analogs with sufficiently improved binding capacity orcross-reactivity can also be tested for immunogenicity inHLA-B7-transgenic mice, following for example, IFA immunization orlipopeptide immunization. Analoged peptides are additionally tested forthe ability to stimulate a recall response using PBMC from patients with108P5H8-expressing tumors.

[0580] Other Analoging Strategies

[0581] Another form of peptide analoging, unrelated to anchor positions,involves the substitution of a cysteine with α-amino butyric acid. Dueto its chemical nature, cysteine has the propensity to form disulfidebridges and sufficiently alter the peptide structurally so as to reducebinding capacity. Substitution of α-amino butyric acid for cysteine notonly alleviates this problem, but has been shown to improve binding andcrossbinding capabilities in some instances (see, e.g., the review bySette et al., In: Persistent Viral Infections, Eds. R. Ahmed and I.Chen, John Wiley & Sons, England, 1999).

[0582] Thus, by the use of single amino acid substitutions, the bindingproperties and/or cross-reactivity of peptide ligands for HLA supertypemolecules can be modulated.

Example 14

[0583] Identification and Confirmation of 108P5H8-Derived Sequences withHLA-DR Binding Motifs

[0584] Peptide epitopes bearing an HLA class II supermotif or motif areidentified and confirmed as outlined below using methodology similar tothat described for HLA Class I peptides.

[0585] Selection of HLA-DR-Supermotif-Bearing Epitopes.

[0586] To identify 108P5H8-derived, HLA class II HTL epitopes, a 108P5H8antigen is analyzed for the presence of sequences bearing anHLA-DR-motif or supermotif. Specifically, 15 -mer sequences are selectedcomprising a DR-supermotif, comprising a 9 -mer core, and three-residueN- and C-terminal flanking regions (15 amino acids total).

[0587] Protocols for predicting peptide binding to DR molecules havebeen developed (Southwood et al., J. Immunol. 160:3363-3373, 1998).These protocols, specific for individual DR molecules, allow thescoring, and ranking, of 9 -mer core regions. Each protocol not onlyscores peptide sequences for the presence of DR-supermotif primaryanchors (i.e., at position 1 and position 6) within a 9 -mer core, butadditionally evaluates sequences for the presence of secondary anchors.Using allele-specific selection tables (see, e.g., Southwood et al.,ibid.), it has been found that these protocols efficiently selectpeptide sequences with a high probability of binding a particular DRmolecule. Additionally, it has been found that performing theseprotocols in tandem, specifically those for DR1, DR4w4, and DR7, canefficiently select DR cross-reactive peptides.

[0588] The 108P5H8-derived peptides identified above are tested fortheir binding capacity for various common HLA-DR molecules. All peptidesare initially tested for binding to the DR molecules in the primarypanel: DR1, DR4w4, and DR7. Peptides binding at least two of these threeDR molecules are then tested for binding to DR2w2 β1, DR2w2 β2, DR6w19,and DR9 molecules in secondary assays. Finally, peptides binding atleast two of the four secondary panel DR molecules, and thuscumulatively at least four of seven different DR molecules, are screenedfor binding to DR4w15, DR5w11, and DR8w2 molecules in tertiary assays.Peptides binding at least seven of the ten DR molecules comprising theprimary, secondary, and tertiary screening assays are consideredcross-reactive DR binders. 108P5H8-derived peptides found to bind commonHLA-DR alleles are of particular interest.

[0589] Selection of DR3 Motif Peptides

[0590] Because HLA-DR3 is an allele that is prevalent in Caucasian,Black, and Hispanic populations, DR3 binding capacity is a relevantcriterion in the selection of HTL epitopes. Thus, peptides shown to becandidates may also be assayed for their DR3 binding capacity. However,in view of the binding specificity of the DR3 motif, peptides bindingonly to DR3 can also be considered as candidates for inclusion in avaccine formulation.

[0591] To efficiently identify peptides that bind DR3, target 108P5H8antigens are analyzed for sequences carrying one of the two DR3-specificbinding motifs reported by Geluk et al. (J. Immunol. 152:5742-5748,1994). The corresponding peptides are then synthesized and confirmed ashaving the ability to bind DR3 with an affinity of 1 μM or better, i.e.,less than 1 μM. Peptides are found that meet this binding criterion andqualify as HLA class II high affinity binders.

[0592] DR3 binding epitopes identified in this manner are included invaccine compositions with DR supermotif-bearing peptide epitopes.

[0593] Similarly to the case of HLA class I motif-bearing peptides, theclass II motif-bearing peptides are analoged to improve affinity orcross-reactivity. For example, aspartic acid at position 4 of the 9 -mercore sequence is an optimal residue for DR3 binding, and substitutionfor that residue often improves DR 3 binding.

Example 15

[0594] Immunogenicity of 108P5H8-Derived HTL Epitopes

[0595] This example determines immunogenic DR supermotif- and DR3motif-bearing epitopes among those identified using the methodology setforth herein.

[0596] Immunogenicity of HTL epitopes are confirmed in a manneranalogous to the determination of immunogenicity of CTL epitopes, byassessing the ability to stimulate HTL responses and/or by usingappropriate transgenic mouse models. Immunogenicity is determined byscreening for: 1.) in vitro primary induction using normal PBMC or 2.)recall responses from patients who have 108P5H8-expressing tumors.

Example 16

[0597] Calculation of Phenotypic Frequencies of HLA-Supertypes inVarious Ethnic Backgrounds to Determine Breadth of Population Coverage

[0598] This example illustrates the assessment of the breadth ofpopulation coverage of a vaccine composition comprised of multipleepitopes comprising multiple supermotifs and/or motifs.

[0599] In order to analyze population coverage, gene frequencies of HLAalleles are determined. Gene frequencies for each HLA allele arecalculated from antigen or allele frequencies utilizing the binomialdistribution formulae gf=1−(SQRT(1−af)) (see, e.g., Sidney et al., HumanImmunol. 45:79-93, 1996). To obtain overall phenotypic frequencies,cumulative gene frequencies are calculated, and the cumulative antigenfrequencies derived by the use of the inverse formula [af=1−(1−Cgf)²].

[0600] Where frequency data is not available at the level of DNA typing,correspondence to the serologically defined antigen frequencies isassumed. To obtain total potential supertype population coverage nolinkage disequilibrium is assumed, and only alleles confirmed to belongto each of the supertypes are included (minimal estimates). Estimates oftotal potential coverage achieved by inter-loci combinations are made byadding to the A coverage the proportion of the non-A covered populationthat could be expected to be covered by the B alleles considered (e.g.,total=A+B*(1−A)). Confirmed members of the A3-like supertype are A3,A11, A31, A*3301,and A*6801. Although the A3-like supertype may alsoinclude A34, A66, and A*7401, these alleles were not included in overallfrequency calculations. Likewise, confirmed members of the A2-likesupertype family are A*0201, A*0202, A*0203, A*0204, A*0205, A*0206,A*0207, A*6802, and A*6901. Finally, the B7-like supertype-confirmedalleles are: B7, B*3501-03, B51, B*5301, B*5401,B*5501-2, B*5601,B*6701, and B*7801 (potentially also B*1401, B*3504-06, B*4201, andB*5602).

[0601] Population coverage achieved by combining the A2-, A3- andB7-supertypes is approximately 86% in five major ethnic groups. Coveragemay be extended by including peptides bearing the A1 and A24 motifs. Onaverage, Al is present in 12% and A24 in 29% of the population acrossfive different major ethnic groups (Caucasian, North American Black,Chinese, Japanese, and Hispanic). Together, these alleles arerepresented with an average frequency of 39% in these same ethnicpopulations. The total coverage across the major ethnicities when A1 andA24 are combined with the coverage of the A2-, A3- and B7-supertypealleles is >95%. An analogous approach can be used to estimatepopulation coverage achieved with combinations of class II motif-bearingepitopes.

[0602] Immunogenicity studies in humans (e.g., Bertoni et al., J. Clin.Invest. 100:503, 1997; Doolan et al., Immunity 7:97, 1997; and Threlkeldet al., J. Immunol. 159:1648, 1997) have shown that highlycross-reactive binding peptides are almost always recognized asepitopes. The use of highly cross-reactive binding peptides is animportant selection criterion in identifying candidate epitopes forinclusion in a vaccine that is immunogenic in a diverse population.

[0603] With a sufficient number of epitopes (as disclosed herein andfrom the art), an average population coverage is predicted to be greaterthan 95% in each of five major ethnic populations. The game theory MonteCarlo simulation analysis, which is known in the art (see e.g., Osborne,M. J. and Rubinstein, A. “A course in game theory” MIT Press, 1994), canbe used to estimate what percentage of the individuals in a populationcomprised of the Caucasian, North American Black, Japanese, Chinese, andHispanic ethnic groups would recognize the vaccine epitopes describedherein. A preferred percentage is 90%. A more preferred percentage is95%.

Example 17

[0604] CTL Recognition of Endogenously Processed Antigens After Priming

[0605] This example confirms that CTL induced by native or analogedpeptide epitopes identified and selected as described herein recognizeendogenously synthesized, i.e., native antigens.

[0606] Effector cells isolated from transgenic mice that are immunizedwith peptide epitopes, for example HLA-A2 supermotif-bearing epitopes,are re-stimulated in vitro using peptide-coated stimulator cells. Sixdays later, effector cells are assayed for cytotoxicity and the celllines that contain peptide-specific cytotoxic activity are furtherre-stimulated. An additional six days later, these cell lines are testedfor cytotoxic activity on 51Cr labeled Jurkat-A2.1/K^(b) target cells inthe absence or presence of peptide, and also tested on ⁵¹Cr labeledtarget cells bearing the endogenously synthesized antigen, i.e. cellsthat are stably transfected with 108P5H8 expression vectors.

[0607] The results demonstrate that CTL lines obtained from animalsprimed with peptide epitope recognize endogenously synthesized 108P5H8antigen. The choice of transgenic mouse model to be used for such ananalysis depends upon the epitope(s) that are being evaluated. Inaddition to HLA-A*0201/K^(b) transgenic mice, several other transgenicmouse models including mice with human A11, which may also be used toevaluate A3 epitopes, and B7 alleles have been characterized and others(e.g., transgenic mice for HLA-A1 and A24) are being developed. HLA-DR1and HLA-DR3 mouse models have also been developed, which may be used toevaluate HTL epitopes.

Example 18

[0608] Activity of CTL-HTL Conjugated Epitopes in Transgenic Mice

[0609] This example illustrates the induction of CTLs and HTLs intransgenic mice, by use of a 108P5H8-derived CTL and HTL peptide vaccinecompositions. The vaccine composition used herein comprise peptides tobe administered to a patient with a 108P5H8-expressing tumor. Thepeptide composition can comprise multiple CTL and/or HTL epitopes. Theepitopes are identified using methodology as described herein. Thisexample also illustrates that enhanced immunogenicity can be achieved byinclusion of one or more HTL epitopes in a CTL vaccine composition; sucha peptide composition can comprise an HTL epitope conjugated to a CTLepitope. The CTL epitope can be one that binds to multiple HLA familymembers at an affinity of 500 nM or less, or analogs of that epitope.The peptides may be lipidated, if desired.

[0610] Immunization procedures. Immunization of transgenic mice isperformed as described (Alexander et al., J. Immunol. 159:4753-4761,1997). For example, A2/K^(b) mice, which are transgenic for the humanHLA A2.1 allele and are used to confirm the immunogenicity of HLA-A*0201motif- or HLA-A2 supermotif-bearing epitopes, and are primedsubcutaneously (base of the tail) with a 0.1 ml of peptide in IncompleteFreund's Adjuvant, or if the peptide composition is a lipidated CTL/HTLconjugate, in DMSO/saline, or if the peptide composition is apolypeptide, in PBS or Incomplete Freund's Adjuvant. Seven days afterpriming, splenocytes obtained from these animals are restimulated withsyngenic irradiated LPS-activated lymphoblasts coated with peptide.

[0611] Cell lines. Target cells for peptide-specific cytotoxicity assaysare Jurkat cells transfected with the HLA-A2.1/K^(b) chimeric gene(e.g., Vitiello et al., J. Exp. Med. 173:1007, 1991)

[0612] In vitro CTL activation: One week after priming, spleen cells(30×10⁶ cells/flask) are co-cultured at 37° C. with syngeneic,irradiated (3000 rads), peptide coated lymphoblasts (10×10⁶ cells/flask)in 10 ml of culture medium/T25 flask. After six days, effector cells areharvested and assayed for cytotoxic activity.

[0613] Assay for cytotoxic activity: Target cells (1.0 to 1.5×10⁶) areincubated at 37° C. in the presence of 200 μl of ⁵¹Cr. After 60 minutes,cells are washed three times and resuspended in R10 medium. Peptide isadded where required at a concentration of 1 μg/ml. For the assay, 10⁴⁵¹Cr-labeled target cells are added to different concentrations ofeffector cells (final volume of 200 μl) in U-bottom 96-well plates.After a six hour incubation period at 37° C., a 0.1 ml aliquot ofsupernatant is removed from each well and radioactivity is determined ina Micromedic automatic gamma counter. The percent specific lysis isdetermined by the formula: percent specific release=100× (experimentalrelease−spontaneous release)/(maximum release−spontaneous release). Tofacilitate comparison between separate CTL assays run under the sameconditions, % ⁵¹Cr release data is expressed as lytic units/10⁶ cells.One lytic unit is arbitrarily defined as the number of effector cellsrequired to achieve 30% lysis of 10,000 target cells in a six hour ⁵¹Crrelease assay. To obtain specific lytic units/10⁶, the lytic units/10⁶obtained in the absence of peptide is subtracted from the lyticunits/10⁶ obtained in the presence of peptide. For example, if 30% ⁵¹Crrelease is obtained at the effector (E): target (T) ratio of 50:1 (i.e.,5×10⁵ effector cells for 10,000 targets) in the absence of peptide and5:1 (i.e., 5×10⁴ effector cells for 10,000 targets) in the presence ofpeptide, the specific lytic units would be: [({fraction(1/50,000)})−({fraction (1/500,000)})]×10⁶=18 LU.

[0614] The results are analyzed to assess the magnitude of the CTLresponses of animals injected with the immunogenic CTL/HTL conjugatevaccine preparation and are compared to the magnitude of the CTLresponse achieved using, for example, CTL epitopes as outlined above inthe Example entitled “Confirmation of Immunogenicity”. Analyses similarto this may be performed to confirm the immunogenicity of peptideconjugates containing multiple CTL epitopes and/or multiple HTLepitopes. In accordance with these procedures, it is found that a CTLresponse is induced, and concomitantly that an HTL response is inducedupon administration of such compositions.

Example 19

[0615] Selection of CTL and HTL Epitopes for Inclusion in an108P5H8-Specific Vaccine.

[0616] This example illustrates a procedure for selecting peptideepitopes for vaccine compositions of the invention. The peptides in thecomposition can be in the form of a nucleic acid sequence, either singleor one or more sequences (i.e., minigene) that encodes peptide(s), orcan be single and/or polyepitopic peptides.

[0617] The following principles are utilized when selecting a pluralityof epitopes for inclusion in a vaccine composition. Each of thefollowing principles is balanced in order to make the selection.

[0618] Epitopes are selected which, upon administration, mimic immuneresponses that are correlated with 108P5H8 clearance. The number ofepitopes used depends on observations of patients who spontaneouslyclear 108P5H8. For example, if it has been observed that patients whospontaneously clear 108P5H8 generate an immune response to at leastthree (3) from 108P5H8 antigen, then three or four (3-4) epitopes shouldbe included for HLA class I. A similar rationale is used to determineHLA class II epitopes.

[0619] Epitopes are often selected that have a binding affinity of anIC₅₀ of 500 nM or less for an HLA class I molecule, or for class II, anIC₅₀ of 1000 nM or less; or HLA Class I peptides with high bindingscores from the BIAS web site, at URL bimas.dcrt.nih.gov/.

[0620] In order to achieve broad coverage of the vaccine through out adiverse population, sufficient supermotif bearing peptides, or asufficient array of allele-specific motif bearing peptides, are selectedto give broad population coverage. In one embodiment, epitopes areselected to provide at least 80% population coverage. A Monte Carloanalysis, a statistical evaluation known in the art, can be employed toassess breadth, or redundancy, of population coverage.

[0621] When creating polyepitopic compositions, or a minigene thatencodes same, it is typically desirable to generate the smallest peptidepossible that encompasses the epitopes of interest. The principlesemployed are similar, if not the same, as those employed when selectinga peptide comprising nested epitopes. For example, a protein sequencefor the vaccine composition is selected because it has maximal number ofepitopes contained within the sequence, i.e., it has a highconcentration of epitopes. Epitopes may be nested or overlapping (i.e.,frame shifted relative to one another). For example, with overlappingepitopes, two 9 -mer epitopes and one 10 -mer epitope can be present ina 10 amino acid peptide. Each epitope can be exposed and bound by an HLAmolecule upon administration of such a peptide. A multi-epitopic,peptide can be generated synthetically, recombinantly, or via cleavagefrom the native source. Alternatively, an analog can be made of thisnative sequence, whereby one or more of the epitopes comprisesubstitutions that alter the cross-reactivity and/or binding affinityproperties of the polyepitopic peptide. Such a vaccine composition isadministered for therapeutic or prophylactic purposes. This embodimentprovides for the possibility that an as yet undiscovered aspect ofimmune system processing will apply to the native nested sequence andthereby facilitate the production of therapeutic or prophylactic immuneresponse-inducing vaccine compositions. Additionally such an embodimentprovides for the possibility of motif-bearing epitopes for an HLA makeupthat is presently unknown. Furthermore, this embodiment (absent thecreating of any analogs) directs the immune response to multiple peptidesequences that are actually present in 108P5H8, thus avoiding the needto evaluate any junctional epitopes. Lastly, the embodiment provides aneconomy of scale when producing nucleic acid vaccine compositions.Related to this embodiment, computer programs can be derived inaccordance with principles in the art, which identify in a targetsequence, the greatest number of epitopes per sequence length.

[0622] A vaccine composition comprised of selected peptides, whenadministered, is safe, efficacious, and elicits an immune responsesimilar in magnitude to an immune response that controls or clears cellsthat bear or overexpress 108P5H8.

Example 20

[0623] Construction of “Minigene” Multi-Epitope DNA Plasmids

[0624] This example discusses the construction of a minigene expressionplasmid. Minigene plasmids may, of course, contain variousconfigurations of B cell, CTL and/or HTL epitopes or epitope analogs asdescribed herein.

[0625] A minigene expression plasmid typically includes multiple CTL andHTL peptide epitopes. In the present example, HLA-A2, -A3, -B7supermotif-bearing peptide epitopes and HLA-A1 and -A24 motif-bearingpeptide epitopes are used in conjunction with DR supermotif-bearingepitopes and/or DR3 epitopes. HLA class I supermotif or motif-bearingpeptide epitopes derived 108P5H8, are selected such that multiplesupermotifs/motifs are represented to ensure broad population coverage.Similarly, HLA class II epitopes are selected from 108P5H8 to providebroad population coverage, i.e. both HLA DR-1-4-7 supermotif-bearingepitopes and HLA DR-3 motif-bearing epitopes are selected for inclusionin the minigene construct. The selected CTL and HTL epitopes are thenincorporated into a minigene for expression in an expression vector.

[0626] Such a construct may additionally include sequences that directthe HTL epitopes to the endoplasmic reticulum. For example, the Iiprotein may be fused to one or more HTL epitopes as described in theart, wherein the CLIP sequence of the Ii protein is removed and replacedwith an HLA class II epitope sequence so that HLA class II epitope isdirected to the endoplasmic reticulum, where the epitope binds to an HLAclass II molecules.

[0627] This example illustrates the methods to be used for constructionof a minigene-bearing expression plasmid. Other expression vectors thatmay be used for minigene compositions are available and known to thoseof skill in the art.

[0628] The minigene DNA plasmid of this example contains a consensusKozak sequence and a consensus murine kappa Ig-light chain signalsequence followed by CTL and/or HTL epitopes selected in accordance withprinciples disclosed herein. The sequence encodes an open reading framefused to the Myc and His antibody epitope tag coded for by the pcDNA 3.1Myc-His vector.

[0629] Overlapping oligonucleotides that can, for example, average about70 nucleotides in length with 15 nucleotide overlaps, are synthesizedand HPLC-purified. The oligonucleotides encode the selected peptideepitopes as well as appropriate linker nucleotides, Kozak sequence, andsignal sequence. The final multiepitope minigene is assembled byextending the overlapping oligonucleotides in three sets of reactionsusing PCR. A Perkin/Elmer 9600 PCR machine is used and a total of 30cycles are performed using the following conditions: 95° C. for 15 sec,annealing temperature (5° below the lowest calculated Tm of each primerpair) for 30 sec, and 72° C. for 1 min.

[0630] For example, a minigene is prepared as follows. For a first PCRreaction, 5 μg of each of two oligonucleotides are annealed andextended: In an example using eight oligonucleotides, i.e., four pairsof primers, oligonucleotides 1+2, 3+4, 5+6, and 7+8 are combined in 100μl reactions containing Pfu polymerase buffer (1×=10 mM KCL, 10 mM(NH4)₂SO₄, 20 mM Tris-chloride, pH 8.75, 2 mM MgSO₄, 0.1% Triton X-100,100 μg/ml BSA), 0.25 mM each dNTP, and 2.5 U of Pfu polymerase. Thefull-length dimer products are gel-purified, and two reactionscontaining the product of 1+2 and 3+4, and the product of 5+6 and 7+8are mixed, annealed, and extended for 10 cycles. Half of the tworeactions are then mixed, and 5 cycles of annealing and extensioncarried out before flanking primers are added to amplify the full lengthproduct. The full-length product is gel-purified and cloned intopCR-blunt (Invitrogen) and individual clones are screened by sequencing.

Example 21

[0631] The Plasmid Construct and the Degree to Which it InducesImmunogenicity.

[0632] The degree to which a plasmid construct, for example a plasmidconstructed in accordance with the previous Example, is able to induceimmunogenicity is confirmed in vitro by determining epitope presentationby APC following transduction or transfection of the APC with anepitope-expressing nucleic acid construct. Such a study determines“antigenicity” and allows the use of human APC. The assay determines theability of the epitope to be presented by the APC in a context that isrecognized by a T cell by quantifying the density of epitope-HLA class Icomplexes on the cell surface. Quantitation can be performed by directlymeasuring the amount of peptide eluted from the APC (see, e.g., Sijts etal., J. Immunol. 156:683-692, 1996; Demotz et al., Nature 342:682-684,1989); or the number of peptide-HLA class I complexes can be estimatedby measuring the amount of lysis or lymphokine release induced bydiseased or transfected target cells, and then determining theconcentration of peptide necessary to obtain. equivalent levels of lysisor lymphokine release (see, e.g., Kageyama et al., J. Immunol.154:567-576, 1995).

[0633] Alternatively, immunogenicity is confirmed through in vivoinjections into mice and subsequent in vitro assessment of CTL and HTLactivity, which are analyzed using cytotoxicity and proliferationassays, respectively, as detailed e.g., in Alexander et al., Immunity1:751-761, 1994.

[0634] For example, to confirm the capacity of a DNA minigene constructcontaining at least one HLA-A2 supermotif peptide to induce CTLs invivo, HLA-A2.1/K^(b) transgenic mice, for example, are immunizedintramuscularly with 100 μg of naked cDNA. As a means of comparing thelevel of CTLs induced by cDNA immunization, a control group of animalsis also immunized with an actual peptide composition that comprisesmultiple epitopes synthesized as a single polypeptide as they would beencoded by the minigene.

[0635] Splenocytes from immunized animals are stimulated twice with eachof the respective compositions (peptide epitopes encoded in the minigeneor the polyepitopic peptide), then assayed for peptide-specificcytotoxic activity in a ⁵¹Cr release assay. The results indicate themagnitude of the CTL response directed against the A2-restrictedepitope, thus indicating the in vivo immunogenicity of the minigenevaccine and polyepitopic vaccine.

[0636] It is, therefore, found that the minigene elicits immuneresponses directed toward the HLA-A2 supermotif peptide epitopes as doesthe polyepitopic peptide vaccine. A similar analysis is also performedusing other HLA-A3 and HLA-B7 transgenic mouse models to assess CTLinduction by HLA-A3 and HLA-B7 motif or supermotif epitopes, whereby itis also found that the minigene elicits appropriate immune responsesdirected toward the provided epitopes.

[0637] To confirm the capacity of a class II epitope-encoding minigeneto induce HTLs in vivo, DR transgenic mice, or for those epitopes thatcross react with the appropriate mouse MHC molecule, I-A^(b)-restrictedmice, for example, are immunized intramuscularly with 100 μg of plasmidDNA. As a means of comparing the level of HTLs induced by DNAimmunization, a group of control animals is also immunized with anactual peptide composition emulsified in complete Freund's adjuvant.CD4+ T cells, i.e. HTLs, are purified from splenocytes of immunizedanimals and stimulated with each of the respective compositions(peptides encoded in the minigene). The HTL response is measured using a³H-thymidine incorporation proliferation assay, (see, e.g., Alexander etal. Immunity 1:751-761, 1994). The results indicate the magnitude of theHTL response, thus demonstrating the in vivo immunogenicity of theminigene.

[0638] DNA minigenes, constructed as described in the previous Example,can also be confirmed as a vaccine in combination with a boosting agentusing a prime boost protocol. The boosting agent can consist ofrecombinant protein (e.g., Barnett et al., Aids Res. and HumanRetroviruses 14, Supplement 3:S299-S309, 1998) or recombinant vaccinia,for example, expressing a minigene or DNA encoding the complete proteinof interest (see, e.g., Hanke et al., Vaccine 16:439-445, 1998; Sedegahet al., Proc. Natl. Acad. Sci USA 95:7648-53,1998; Hanke and McMichael,Immunol. Letters 66:177-181, 1999; and Robinson et al., Nature Med.5:526-34, 1999).

[0639] For example, the efficacy of the DNA minigene used in a primeboost protocol is initially evaluated in transgenic mice. In thisexample, A2.1/K^(b) transgenic mice are immunized IM with 100 μg of aDNA minigene encoding the immunogenic peptides including at least oneHLA-A2 supermotif-bearing peptide. After an incubation period (rangingfrom 3-9 weeks), the mice are boosted IP with 10⁷ pfu/mouse of arecombinant vaccinia virus expressing the same sequence encoded by theDNA minigene. Control mice are immunized with 100 μg of DNA orrecombinant vaccinia without the minigene sequence, or with DNA encodingthe minigene, but without the vaccinia boost. After an additionalincubation period of two weeks, splenocytes from the mice areimmediately assayed for peptide-specific activity in an ELISPOT assay.Additionally, splenocytes are stimulated in vitro with the A2-restrictedpeptide epitopes encoded in the minigene and recombinant vaccinia, thenassayed for peptide-specific activity in an alpha, beta and/or gamma IFNELISA.

[0640] It is found that the minigene utilized in a prime-boost protocolelicits greater immune responses toward the HLA-A2 supermotif peptidesthan with DNA alone. Such an analysis can also be performed usingHLA-A11 or HLA-B7 transgenic mouse models to assess CTL induction byHLA-A3 or HLA-B7 motif or supermotif epitopes. The use of prime boostprotocols in humans is described below in the Example entitled“Induction of CTL Responses Using a Prime Boost Protocol.”

Example 22

[0641] Peptide Composition for Prophylactic Uses

[0642] Vaccine compositions of the present invention can be used toprevent 108P5H8 expression in persons who are at risk for tumors thatbear this antigen. For example, a polyepitopic peptide epitopecomposition (or a nucleic acid comprising the same) containing multipleCTL and HTL epitopes such as those selected in the above Examples, whichare also selected to target greater than 80% of the population, isadministered to individuals at risk for a 108P5H8-associated tumor.

[0643] For example, a peptide-based composition is provided as a singlepolypeptide that encompasses multiple epitopes. The vaccine is typicallyadministered in a physiological solution that comprises an adjuvant,such as Incomplete Freunds Adjuvant. The dose of peptide for the initialimmunization is from about 1 to about 50,000 μg, generally 100-5,000 μg,for a 70 kg patient. The initial administration of vaccine is followedby booster dosages at 4 weeks followed by evaluation of the magnitude ofthe immune response in the patient, by techniques that determine thepresence of epitope-specific CTL populations in a PBMC sample.Additional booster doses are administered as required. The compositionis found to be both safe and efficacious as a prophylaxis against108P5H8-associated disease.

[0644] Alternatively, a composition typically comprising transfectingagents is used for the administration of a nucleic acid-based vaccine inaccordance with methodologies known in the art and disclosed herein.

Example 23

[0645] Polyepitopic Vaccine Compositions Derived from Native 108P5H8Sequences

[0646] A native 108P5H8 polyprotein sequence is analyzed, preferablyusing computer algorithms defined for each class I and/or class IIsupermotif or motif, to identify “relatively short” regions of thepolyprotein that comprise multiple epitopes. The “relatively short”regions are preferably less in length than an entire native antigen.This relatively short sequence that contains multiple distinct oroverlapping, “nested” epitopes is selected; it can be used to generate aminigene construct. The construct is engineered to express the peptide,which corresponds to the native protein sequence. The “relatively short”peptide is generally less than 250 amino acids in length, often lessthan 100 amino acids in length, preferably less than 75 amino acids inlength, and more preferably less than 50 amino acids in length. Theprotein sequence of the vaccine composition is selected because it hasmaximal number of epitopes contained within the sequence, i.e., it has ahigh concentration of epitopes. As noted herein, epitope motifs may benested or overlapping (i.e., frame shifted relative to one another). Forexample, with overlapping epitopes, two 9 -mer epitopes and one 10 -merepitope can be present in a 10 amino acid peptide. Such a vaccinecomposition is administered for therapeutic or prophylactic purposes.

[0647] The vaccine composition will include, for example, multiple CTLepitopes from 108P5H8 antigen and at least one HTL epitope. Thispolyepitopic native sequence is administered either as a peptide or as anucleic acid sequence which encodes the peptide. Alternatively, ananalog can be made of this native sequence, whereby one or more of theepitopes comprise substitutions that alter the cross-reactivity and/orbinding affinity properties of the polyepitopic peptide.

[0648] The embodiment of this example provides for the possibility thatan as yet undiscovered aspect of immune system processing will apply tothe native nested sequence and thereby facilitate the production oftherapeutic or prophylactic immune response-inducing vaccinecompositions. Additionally such an embodiment provides for thepossibility of motif-bearing epitopes for an HLA makeup that ispresently unknown. Furthermore, this embodiment (excluding an analogedembodiment) directs the immune response to multiple peptide sequencesthat are actually present in native 108P5H8, thus avoiding the need toevaluate any junctional epitopes. Lastly, the embodiment provides aneconomy of scale when producing peptide or nucleic acid vaccinecompositions.

[0649] Related to this embodiment, computer programs are available inthe art which can be used to identify in a target sequence, the greatestnumber of epitopes per sequence length.

Example 24

[0650] Polyepitopic Vaccine Compositions from Multiple Antigens

[0651] The 108P5H8 peptide epitopes of the present invention are used inconjunction with epitopes from other target tumor-associated antigens,to create a vaccine composition that is useful for the prevention ortreatment of cancer that expresses 108P5H8 and such other antigens. Forexample, a vaccine composition can be provided as a single polypeptidethat incorporates multiple epitopes from 108P5H8 as well astumor-associated antigens that are often expressed with a target cancerassociated with 108P5H8 expression, or can be administered as acomposition comprising a cocktail of one or more discrete epitopes.Alternatively, the vaccine can be administered as a minigene constructor as dendritic cells which have been loaded with the peptide epitopesin vitro.

Example 25

[0652] Use of Peptides to Evaluate an Immune Response

[0653] Peptides of the invention may be used to analyze an immuneresponse for the presence of specific antibodies, CTL or HTL directed to108P5H8. Such an analysis can be performed in a manner described by Ogget al., Science 279:2103-2106, 1998. In this Example, peptides inaccordance with the invention are used as a reagent for diagnostic orprognostic purposes, not as an immunogen.

[0654] In this example highly sensitive human leukocyte antigentetrameric complexes (“tetramers”) are used for a cross-sectionalanalysis of, for example, 108P5H8 HLA-A*0201-specific CTL frequenciesfrom HLA A*0201-positive individuals at different stages of disease orfollowing immunization comprising an 168P5H8 peptide containing anA*0201 motif. Tetrameric complexes are synthesized as described (Museyet al., N. Engl. J. Med. 337:1267, 1997). Briefly, purified HLA heavychain (A*0201 in this example) and β2-microglobulin are synthesized bymeans of a prokaryotic expression system. The heavy chain is modified bydeletion of the transmembrane-cytosolic tail and COOH-terminal additionof a sequence containing a BirA enzymatic biotinylation site. The heavychain, β2-microglobulin, and peptide are refolded by dilution. The 45-kD refolded product is isolated by fast protein liquid chromatographyand then biotinylated by BirA in the presence of biotin (Sigma, St.Louis, Mo.), adenosine 5′ triphosphate and magnesium.Streptavidin-phycoerythrin conjugate is added in a 1:4 molar ratio, andthe tetrameric product is concentrated to 1 mg/ml. The resulting productis referred to as tetramer-phycoerythrin.

[0655] For the analysis of patient blood samples, approximately onemillion PBMCs are centrifuged at 300 g for 5 minutes and resuspended in50 μl of cold phosphate-buffered saline. Tri-color analysis is performedwith the tetramer-phycoerythrin, along with anti-CD8-Tricolor, andanti-CD38. The PBMCs are incubated with tetramer and antibodies on icefor 36 to 60 min and then washed twice before formaldehyde fixation.Gates are applied to contain >99.98% of control samples. Controls forthe tetramers include both A*0201-negative individuals andA*0201-positive non-diseased donors. The percentage of cells stainedwith the tetramer is then determined by flow cytometry. The resultsindicate the number of cells in the PBMC sample that containepitope-restricted CTLs, thereby readily indicating the extent of immuneresponse to the 108P5H8 epitope, and thus the status of exposure to108P5H8, or exposure to a vaccine that elicits a protective ortherapeutic response.

Example 26

[0656] Use of Peptide Epitopes to Evaluate Recall Responses

[0657] The peptide epitopes of the invention are used as reagents toevaluate T cell responses, such as acute or recall responses, inpatients. Such an analysis may be performed on patients who haverecovered from 108P5H8-associated disease or who have been vaccinatedwith an 108P5H8 vaccine.

[0658] For example, the class I restricted CTL response of persons whohave been vaccinated may be analyzed. The vaccine may be any 108P5H8vaccine. PBMC are collected from vaccinated individuals and HLA typed.Appropriate peptide epitopes of the invention that, optimally, bearsupermotifs to provide cross-reactivity with multiple HLA supertypefamily members, are then used for analysis of samples derived fromindividuals who bear that HLA type.

[0659] PBMC from vaccinated individuals are separated onFicoll-Histopaque density gradients (Sigma Chemical Co., St. Louis,Mo.), washed three times in HBSS (GIBCO Laboratories), resuspended inRPMI-1640 (GIBCO Laboratories) supplemented with L-glutamine (2 mM),penicillin (50 U/ml), streptomycin (50 μg/ml), and Hepes (10 mM)containing 10% heat-inactivated human AB serum (complete RPMI) andplated using microculture formats. A synthetic peptide comprising anepitope of the invention is added at 10 μg/ml to each well and HBV core128-140 epitope is added at 1 μg/ml to each well as a source of T cellhelp during the first week of stimulation.

[0660] In the microculture format, 4×10⁵ PBMC are stimulated withpeptide in 8 replicate cultures in 96-well round bottom plate in 100μl/well of complete RPMI. On days 3 and 10, 100 μl of complete RPMI and20 U/ml final concentration of rIL-2 are added to each well. On day 7the cultures are transferred into a 96-well flat-bottom plate andrestimulated with peptide, rIL-2 and 10⁵ irradiated (3,000 rad)autologous feeder cells. The cultures are tested for cytotoxic activityon day 14. A positive CTL response requires two or more of the eightreplicate cultures to display greater than 10% specific ⁵¹Cr release,based on comparison with non-diseased control subjects as previouslydescribed (Rehermarm, et al., Nature Med. 2:1104,1108, 1996; Rehermannet al., J. Clin. Invest. 97:1655-1665, 1996; and Rehermann et al. J.Clin. Invest. 98:1432-1440, 1996).

[0661] Target cell lines are autologous and allogeneic EBV-transformedB-LCL that are either purchased from the American Society forHistocompatibility and Immunogenetics (ASHI, Boston, Mass.) orestablished from the pool of patients as described (Guilhot, et al. J.Virol. 66:2670-2678, 1992).

[0662] Cytotoxicity assays are performed in the following manner. Targetcells consist of either allogeneic HLA-matched or autologousEBV-transformed B lymphoblastoid cell line that are incubated overnightwith the synthetic peptide epitope of the invention at 10 μM, andlabeled with 100 μCi of ⁵¹Cr (Amersham Corp., Arlington Heights, Ill.)for 1 hour after which they are washed four times with HBSS.

[0663] Cytolytic activity is determined in a standard 4 -h, split well⁵¹Cr release assay using U-bottomed 96 well plates containing 3,000targets/well. Stimulated PBMC are tested at effector/target (E/T) ratiosof 20-50:1 on day 14. Percent cytotoxicity is determined from theformula: 100× [(experimental release−spontaneous release)/maximumrelease−spontaneous release)]. Maximum release is determined by lysis oftargets by detergent (2% Triton X-100; Sigma Chemical Co., St. Louis,Mo.). Spontaneous release is <25% of maximum release for allexperiments.

[0664] The results of such an analysis indicate the extent to whichHLA-restricted CTL populations have been stimulated by previous exposureto 108P5H8 or an 108P5H8 vaccine.

[0665] Similarly, Class II restricted HTL responses may also beanalyzed. Purified PBMC are cultured in a 96-well flat bottom plate at adensity of 1.5×10⁵ cells/well and are stimulated with 10 μg/ml syntheticpeptide of the invention, whole 108P5H8 antigen, or PHA. Cells areroutinely plated in replicates of 4-6 wells for each condition. Afterseven days of culture, the medium is removed and replaced with freshmedium containing 10 U/ml IL-2. Two days later, 1 μCi ³H-thymidine isadded to each well and incubation is continued for an additional 18hours. Cellular DNA is then harvested on glass fiber mats and analyzedfor ³H-thymidine incorporation. Antigen-specific T cell proliferation iscalculated as the ratio of ³H-thymidine incorporation in the presence ofantigen divided by the ³H-thymidine incorporation in the absence ofantigen.

Example 27

[0666] Induction of Specific CTL Response in Humans

[0667] A human clinical trial for an immunogenic composition comprisingCTL and HTL epitopes of the invention is set up as an IND Phase I, doseescalation study and carried out as a randomized, double-blind,placebo-controlled trial. Such a trial is designed, for example, asfollows:

[0668] A total of about 27 individuals are enrolled and divided into 3groups:

[0669] Group I: 3 subjects are injected with placebo and 6 subjects areinjected with 5 μg of peptide composition;

[0670] Group II: 3 subjects are injected with placebo and 6 subjects areinjected with 50 μg peptide composition;

[0671] Group III: 3 subjects are injected with placebo and 6 subjectsare injected with 500 μg of peptide composition.

[0672] After 4 weeks following the first injection, all subjects receivea booster inoculation at the same dosage.

[0673] The endpoints measured in this study relate to the safety andtolerability of the peptide composition as well as its immunogenicity.Cellular immune responses to the peptide composition are an index of theintrinsic activity of this the peptide composition, and can therefore beviewed as a measure of biological efficacy. The following summarize theclinical and laboratory data that relate to safety and efficacyendpoints.

[0674] Safety: The incidence of adverse events is monitored in theplacebo and drug treatment group and assessed in terms of degree andreversibility.

[0675] Evaluation of Vaccine Efficacy: For evaluation of vaccineefficacy, subjects are bled before and after injection. Peripheral bloodmononuclear cells are isolated from fresh heparinized blood byFicoll-Hypaque density gradient centrifugation, aliquoted in freezingmedia and stored frozen. Samples are assayed for CTL and HTL activity.

[0676] The vaccine is found to be both safe and efficacious.

Example 28

[0677] Phase II Trials in Patients Expressing 108P5H8

[0678] Phase II trials are performed to study the effect ofadministering the CTL-HTL peptide compositions to patients having cancerthat expresses 108P5H8. The main objectives of the trial are todetermine an effective dose and regimen for inducing CTLs in cancerpatients that express 108P5H8, to establish the safety of inducing a CTLand HTL response in these patients, and to see to what extent activationof CTLs improves the clinical picture of these patients, as manifested,e.g., by the reduction and/or shrinking of lesions. Such a study isdesigned, for example, as follows:

[0679] The studies are performed in multiple centers. The trial designis an open-label, uncontrolled, dose escalation protocol wherein thepeptide composition is administered as a single dose followed six weekslater by a single booster shot of the same dose. The dosages are 50, 500and 5,000 micrograms per injection. Drug-associated adverse effects(severity and reversibility) are recorded.

[0680] There are three patient groupings. The first group is injectedwith 50 micrograms of the peptide composition and the second and thirdgroups with 500 and 5,000 micrograms of peptide composition,respectively. The patients within each group range in age from 21-65 andrepresent diverse ethnic backgrounds. All of them have a tumor thatexpresses 108P5H8.

[0681] Clinical manifestations or antigen-specific T-cell responses aremonitored to assess the effects of administering the peptidecompositions. The vaccine composition is found to be both safe andefficacious in the treatment of 108P5H8-associated disease.

Example 29

[0682] Induction of CTL Responses Using a Prime Boost Protocol

[0683] A prime boost protocol similar in its underlying principle tothat used to confirm the efficacy of a DNA vaccine in transgenic mice,such as described above in the Example entitled “The Plasmid Constructand the Degree to Which It Induces Immunogenicity,” can also be used forthe administration of the vaccine to humans. Such a vaccine regimen caninclude an initial administration of, for example, naked DNA followed bya boost using recombinant virus encoding the vaccine, or recombinantprotein/polypeptide or a peptide mixture administered in an adjuvant.

[0684] For example, the initial immunization may be performed using anexpression vector, such as that constructed in the Example entitled“Construction of ‘Minigene’ Multi-Epitope DNA Plasmids” in the form ofnaked nucleic acid administered IM (or SC or ID) in the amounts of 0.5-5mg at multiple sites. The nucleic acid (0.1 to 1000 μg) can also beadministered using a gene gun. Following an incubation period of 3-4weeks, a booster dose is then administered. The booster can berecombinant fowlpox virus administered at a dose of 5-10⁷ to 5×10⁹ pfu.An alternative recombinant virus, such as an MVA, canarypox, adenovirus,or adeno-associated virus, can also be used for the booster, or thepolyepitopic protein or a mixture of the peptides can be administered.For evaluation of vaccine efficacy, patient blood samples are obtainedbefore immunization as well as at intervals following administration ofthe initial vaccine and booster doses of the vaccine. Peripheral bloodmononuclear cells are isolated from fresh heparinized blood byFicoll-Hypaque density gradient centrifugation, aliquoted in freezingmedia and stored frozen. Samples are assayed for CTL and HTL activity.

[0685] Analysis of the results indicates that a magnitude of responsesufficient to achieve a therapeutic or protective immunity against108P5H8 is generated.

Example 30

[0686] Administration of Vaccine Compositions Using Dendritic Cells (DC)

[0687] Vaccines comprising peptide epitopes of the invention can beadministered using APCs, or “professional” APCs such as DC. In thisexample, peptide-pulsed DC are administered to a patient to stimulate aCTL response in vivo. In this method, dendritic cells are isolated,expanded, and pulsed with a vaccine comprising peptide CTL and HTLepitopes of the invention. The dendritic cells are infused back into thepatient to elicit CTL and HTL responses in vivo. The induced CTL and HTLthen destroy or facilitate destruction, respectively, of the targetcells that bear the 108P5H8 protein from which the epitopes in thevaccine are derived.

[0688] For example, a cocktail of epitope-comprising peptides isadministered ex vivo to PBMC, or isolated DC therefrom. A pharmaceuticalto facilitate harvesting of DC can be used, such as Progenipoietin™(Monsanto, St. Louis, Mo.) or GM-CSF/IL-4. After pulsing the DC withpeptides, and prior to reinfusion into patients, the DC are washed toremove unbound peptides.

[0689] As appreciated clinically, and readily determined by one of skillbased on clinical outcomes, the number of DC reinfused into the patientcan vary (see, e.g., Nature Med. 4:328, 1998; Nature Med. 2:52, 1996 andProstate 32:272, 1997). Although 2-50×10⁶ DC per patient are typicallyadministered, larger number of DC, such as 10⁷ or 10⁸ can also beprovided. Such cell populations typically contain between 50-90% DC.

[0690] In some embodiments, peptide-loaded PBMC are injected intopatients without purification of the DC. For example, PBMC generatedafter treatment with an agent such as Progenipoietin™ are injected intopatients without purification of the DC. The total number of PBMC thatare administered often ranges from 10⁸ to 10¹⁰. Generally, the celldoses injected into patients is based on the percentage of DC in theblood of each patient, as determined, for example, by immunofluorescenceanalysis with specific anti-DC antibodies. Thus, for example, ifProgenipoietin™ mobilizes 2% DC in the peripheral blood of a givenpatient, and that patient is to receive 5×10⁶ DC, then the patient willbe injected with a total of 2.5×10⁸ peptide-loaded PBMC. The percent DCmobilized by an agent such as Progenipoietin™ is typically estimated tobe between 2-10%, but can vary as appreciated by one of skill in theart.

[0691] Ex Vivo Activation of CTL/HTL Responses

[0692] Alternatively, ex vivo CTL or HTL responses to 108P5H8 antigenscan be induced by incubating, in tissue culture, the patient's, orgenetically compatible, CTL or HTL precursor cells together with asource of APC, such as DC, and immunogenic peptides. After anappropriate incubation time (typically about 7-28 days), in which theprecursor cells are activated and expanded into effector cells, thecells are infused into the patient, where they will destroy (CTL) orfacilitate destruction (HTL) of their specific target cells, i.e., tumorcells.

Example 31

[0693] An Alternative Method of Identifying and Confirming Motif-BearingPeptides

[0694] Another method of identifying and confirming motif-bearingpeptides is to elute them from cells bearing defined MHC molecules. Forexample, EBV transformed B cell lines used for tissue typing have beenextensively characterized to determine which HLA molecules they express.In certain cases these cells express only a single type of HLA molecule.These cells can be transfected with nucleic acids that express theantigen of interest, e.g. 108P5H8. Peptides produced by endogenousantigen processing of peptides produced as a result of transfection willthen bind to HLA molecules within the cell and be transported anddisplayed on the cell's surface. Peptides are then eluted from the HLAmolecules by exposure to mild acid conditions and their amino acidsequence determined, e.g., by mass spectral analysis (e.g., Kubo et al.,J. Immunol. 152:3913, 1994). Because the majority of peptides that binda particular HLA molecule are motif-bearing, this is an alternativemodality for obtaining the motif-bearing peptides correlated with theparticular HLA molecule expressed on the cell.

[0695] Alternatively, cell lines that do not express endogenous HLAmolecules can be transfected with an expression construct encoding asingle HLA allele. These cells can then be used as described, i.e., theycan then be transfected with nucleic acids that encode 108P5H8 toisolate peptides corresponding to 108P5H8 that have been presented onthe cell surface. Peptides obtained from such an analysis will bearmotif(s) that correspond to binding to the single HLA allele that isexpressed in the cell.

[0696] As appreciated by one in the art, one can perform a similaranalysis on a cell bearing more than one HLA allele and subsequentlydetermine peptides specific for each HLA allele expressed. Moreover, oneof skill would also recognize that means other than transfection, suchas loading with a protein antigen, can be used to provide a source ofantigen to the cell.

Example 32

[0697] Complementary Polynucleotides

[0698] Sequences complementary to the 108P5H8-encoding sequences, or anyparts thereof, are used to detect, decrease, or inhibit expression ofnaturally occurring 108P5H8. Although use of oligonucleotides comprisingfrom about 15 to 30 base pairs is described, essentially the sameprocedure is used with smaller or with larger sequence fragments.Appropriate oligonucleotides are designed using, e.g., OLIGO 4.06software (National Biosciences) and the coding sequence of 108P5H8. Toinhibit transcription, a complementary oligonucleotide is designed fromthe most unique 5′ sequence and used to prevent promoter binding to thecoding sequence. To inhibit translation, a complementary oligonucleotideis designed to prevent ribosomal binding to a 108P5H8-encodingtranscript.

Example 33

[0699] Purification of Naturally-Occurring or Recombinant 108P5H8 Using108P5H8 Specific Antibodies

[0700] Naturally occurring or recombinant 108P5H8 is substantiallypurified by immunoaffinity chromatography using antibodies specific for108P5H8. An immunoaffinity column is constructed by covalently couplinganti-108P5H8 antibody to an activated chromatographic resin, such asCNBr-activated SEPHAROSE (Amersham Pharmacia Biotech). After thecoupling, the resin is blocked and washed according to themanufacturer's instructions.

[0701] Media containing 108P5H8 are passed over the immunoaffinitycolumn, and the column is washed under conditions that allow thepreferential absorbance of 108P5H8 (e.g., high ionic strength buffers inthe presence of detergent). The column is eluted under conditions thatdisrupt antibody/108P5H8 binding (e.g., a buffer of pH 2 to pH 3, or ahigh concentration of a chaotrope, such as urea or thiocyanate ion), andGCR.P is collected.

Example 34

[0702] Identification of Molecules which Interact with 108P5H8

[0703] 108P5H8, or biologically active fragments thereof, are labeledwith 121 1 Bolton-Hunter reagent. (See, e.g., Bolton et al. (1973)Biochem. J. 133:529.) Candidate molecules previously arrayed in thewells of a multi-well plate are incubated with the labeled 108P5H8,washed, and any wells with labeled 108P5H8 complex are assayed. Dataobtained using different concentrations of 108P5H8 are used to calculatevalues for the number, affinity, and association of 108P5H8 with thecandidate molecules.

Example 35

[0704] In Vivo Assay for 108P5H8 Tumor Growth Promotion

[0705] The effect of a 108P5H8 protein on tumor cell growth can beconfirmed in vivo by gene overexpression in a variety of cancer cellssuch as those in Table I, including prostate, kidney, colon and bladder.For example, SCID mice can be injected SQ on each flank with 1×10⁶prostate, kidney, colon or bladder cancer cells (such as PC3, DU145,SCaBER, UM-UC-3, HT1376, SK-CO, Caco, RT4, T24, Caki, A-498 and SW839cells) containing tkNeo empty vector or 108P5H8.

[0706] At least two strategies can be used:

[0707] (1) Constitutive 108P5H8 expression under regulation of apromoter such as a constitutive promoter obtained from the genomes ofviruses such as polyoma virus, fowlpox virus (UK 2,211,504 publishedJul. 5, 1989), adenovirus (such as Adenovirus 2), bovine papillomavirus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-Bvirus and Simian Virus 40 (SV40), or from heterologous mammalianpromoters, e.g., the actin promoter or an immunoglobulin promoter,provided such promoters are compatible with the host cell systems.

[0708] (2) Regulated expression under control of an inducible vectorsystem, such as ecdysone, tet, etc., can be used provided such promotersare compatible with the host cell systems. Tumor volume is thenmonitored at the appearance of palpable tumors or by following serummarkers such as PSA. Tumor development is followed over time to validatethat 108P5H8-expressing cells grow at a faster rate and/or that tumorsproduced by 108P5H8-expressing cells demonstrate characteristics ofaltered aggressiveness (e.g., enhanced metastasis, vascularization,reduced responsiveness to chemotherapeutic drugs). Tumor volume isevaluated by caliper measurements. Additionally, mice can be implantedwith the same cells orthotopically in the prostate, bladder, colon orkidney to determine if 108P5H8 has an effect on local growth, e.g., inthe prostate, bladder, colon or kidney or on the ability of the cells tometastasize, specifically to lungs or lymph nodes (Saffran et al., ProcNatl Acad Sci U S A. 2001, 98: 2658; Fu, X., et al., Int. J. Cancer,1991. 49: 938-939; Chang, S., et al, Anticancer Res., 1997, 17:3239-3242; Peralta, E. A., et al., J. Urol., 1999. 162: 1806-1811). Forinstance, the orthotopic growth of PC3 and PC3-108P5H8 can be comparedin the prostate of SCID mice. Such experiments reveal the effect of108P5H8 on orthotopic tumor growth, metastasis and/or angiogenicpotential.

[0709] Furthermore, this assay is useful to confirm the inhibitoryeffect of candidate therapeutic compositions, such as for example,108P5H8 antibodies or intrabodies, and 108P5H8 antisense molecules orribozymes, or 108P5H8 directed small molecules, on cells that express a108P5H8 protein.

Example 36

[0710] 108P5H8 Monoclonal Antibody-Mediated Inhibition of HumanXenograft Tumors in Vivo

[0711] The significant expression of 108P5H8, in cancer tissues,together with its restricted expression in normal tissues along with itscell surface expression makes 108P5H8 an excellent target for antibodytherapy. Similarly, 108P5H8 is a target for T cell-based immunotherapy.Thus, the therapeutic efficacy of anti-108P5H8 mAbs is evaluated, e.g.,in human prostate cancer xenograft mouse models usingandrogen-independent LAPC-4 and LAPC-9 xenografts (Craft, N., et al.,Cancer Res, 1999. 59(19): p. 5030-5036), prostate cancer cell linestransfected with 108P5H8 (such as PC3-108P5H8, DU145-108P5H8), in humankidney cancer xenografts (AGS-K3, AGS-K6),.kidney cancer metastases tolymph node (AGS-K6 met) xenografts, and kidney cancer cell linestransfected with 108P5H8 (769P-108P5H8, A498-108P5H8).

[0712] Antibody efficacy on tumor growth and metastasis formation isstudied, e.g., in mouse subcutaneous or orthotopic prostate cancerxenograft models and mouse kidney xenograft models. The antibodies canbe unconjugated, as discussed in this Example, or can be conjugated to atherapeutic modality, as appreciated in the art. Anti-108P5H8 mAbsinhibit formation of both the androgen-dependent LAPC-9 andandrogen-independent PC3-108P5H8 tumor xenografts. Anti-108P5H8 mAbsalso retard the growth of established orthotopic tumors and prolongedsurvival of tumor-bearing mice. These results indicate the utility ofanti-108P5H8 mAbs in the treatment of local and advanced stages of,e.g., prostate cancer. (See, e.g., Saffran, D., et al., PNAS 10:1073-1078 or www.pnas.org/cgi/doi/10.1073/pnas.051624698). These resultsindicate the use of anti-108P5H8 mAbs in the treatment of prostatecancer.

[0713] Administration of the anti-108P5H8 mAbs leads to retardation ofestablished orthotopic tumor growth and inhibition of metastasis todistant sites, resulting in a significant prolongation in the survivalof tumor-bearing mice. These studies indicate that 108P5H8 is anattractive target for immunotherapy and demonstrate the therapeutic useof anti-108P5H8 mAbs for the treatment of local and metastatic cancer.This example demonstrates that unconjugated 108P5H8 monoclonalantibodies are effective to inhibit the growth of human prostate tumorxenografts and human kidney xenografts grown in SCID mice.

[0714] Tumor Inhibition Using Multiple Unconjugated 108P5H8 mAbs

[0715] Materials and Methods

[0716] 108P5H8 Monoclonal Antibodies:

[0717] Monoclonal antibodies are obtained against 108P5H8, such asdescribed in the Example entitled “Generation of 108P5H8 MonoclonalAntibodies (mAbs)” or may be obtained commercially. The antibodies arecharacterized by ELISA, Western blot, FACS, and immunoprecipitation fortheir capacity to bind 108P5H8. Epitope mapping data for theanti-108P5H8 mAbs, as determined by ELISA and Western analysis,recognize epitopes on a 108P5H8 protein. Immunohistochemical analysis ofcancer tissues and cells is performed with these antibodies.

[0718] The monoclonal antibodies are purified from ascites or hybridomatissue culture supernatants by Protein-G Sepharose chromatography,dialyzed against PBS, filter sterilized, and stored at −20° C. Proteindeterminations are performed by a Bradford assay (Bio-Rad, Hercules,Calif.). A therapeutic monoclonal antibody or a cocktail comprising amixture of individual monoclonal antibodies is prepared and used for thetreatment of mice receiving subcutaneous or orthotopic injections of,e.g., LAPC-9 prostate tumor xenografts.

[0719] Cancer Xenografts and Cell Lines

[0720] The LAPC-9 xenograft, which expresses a wild-type androgenreceptor and produces prostate-specific antigen (PSA), is passaged in 6-to 8-week-old male ICR-severe combined immunodeficient (SCID) mice(Taconic Farms) by s.c. trocar implant (Craft, N., et al., 1999, CancerRes. 59:5030-5036). Single-cell suspensions of tumor cells are preparedas described in Craft, et al. The prostate carcinoma cell lines PC3 andDU145 (American Type Culture Collection) are maintained in RPMIsupplemented with L-glutamine and 10% FBS, and the kidney carcinoma lineA498 (American Type Culture Collection) is maintained in DMEMsupplemented with L-glutamine and 10% FBS.

[0721] PC3-108P5H8, DU145-108P5H8 and A498-108P5H8 cell populations aregenerated by retroviral gene transfer as described in Hubert, R. S., etal., STEAP: A Prostate-specific Cell-surface Antigen Highly Expressed inHuman Prostate Tumors, Proc Natl Acad Sci U S A, 1999. 96(25): p.14523-14528. Anti-108P5H8 staining is detected by using, e.g., anFITC-conjugated goat anti-mouse antibody (Southern BiotechnologyAssociates) followed by analysis on a Coulter Epics-XL flow cytometer.

[0722] Xenograft Mouse Models.

[0723] Subcutaneous (s.c.) tumors are generated by injection of 1×10⁶LAPC-9, PC3, PC3-108P5H8, DU145 or DU145-108P5H8 cells mixed at a 1:1dilution with Matrigel (Collaborative Research) in the right flank ofmale SCID mice. To test antibody efficacy on tumor formation, i.p.antibody injections are started on the same day as tumor-cellinjections. As a control, mice are injected with either purified mouseIgG (ICN) or PBS; or a purified monoclonal antibody that recognizes anirrelevant antigen not expressed in human cells. In preliminary studies,no difference is found between mouse IgG or PBS on tumor growth. Tumorsizes are determined by vernier caliper measurements, and the tumorvolume is calculated as length×width×height. Mice with s.c. tumorsgreater than 1.5 cm in diameter are sacrificed. PSA levels aredetermined by using a PSA ELISA kit (Anogen, Mississauga, Ontario).Circulating levels of anti-108P5H8 mAbs are determined by a captureELISA kit (Bethyl Laboratories, Montgomery, Tex.). (See, e.g., (Saffran,D., et al., PNAS 10:1073-1078 orwww.pnas.org/cgi/doi/10.1073/pnas.051624698)

[0724] Orthotopic prostate injections are performed under anesthesia byusing ketamine/xylazine. For prostate orthotopic studies, an incision ismade through the abdominal muscles to expose the bladder and seminalvesicles, which then are delivered through the incision to expose thedorsal prostate. LAPC-9 cells (5×10⁵ ) mixed with Matrigel are injectedinto each dorsal lobe in a 10 μl volume. To monitor tumor growth, miceare bled on a weekly basis for determination of PSA levels. The mice aresegregated into groups for appropriate treatments, with anti-108P5H8 orcontrol mAbs being injected i.p.

[0725] Anti-108P5H8 mAbs Inhibit Growth of 108P5H8-ExpressingXenograft-Cancer Tumors

[0726] The effect of anti-108P5H8 mAbs on tumor formation is tested byusing orthotopic models, e.g., LAPC-9 orthotopic models. As comparedwith the s.c. tumor model, the orthotopic model, which requiresinjection of tumor cells directly in the mouse prostate or kidney,respectively, results in a local tumor growth, development of metastasisin distal sites, deterioration of mouse health, and subsequent death(Saffran, D., et al., PNAS supra; Fu, X., et al., Int J Cancer, 1992.52(6): p. 987-90; Kubota, T., J Cell Biochem, 1994. 56(1): p. 4-8). Thefeatures make the orthotopic model more representative of human diseaseprogression and allow for tracking of the therapeutic effect of mAbs onclinically relevant end points.

[0727] Accordingly, tumor cells are injected into the mouse prostate orkidney, and the mice are segregated into two groups and treated witheither: a) 200-500 μg, of anti-108P5H8 Ab, b) PBS or c) controlnon-specific monoclonal antibody for two to five weeks.

[0728] As noted, a major advantage of the orthotopic prostate-cancermodel is the ability to study the development of metastases. Formationof metastasis in mice bearing established orthotopic tumors is studiedby IHC analysis on lung sections using an antibody against aprostate-specific cell-surface protein STEAP expressed at high levels inLAPC-9 xenografts (Hubert, R. S., et al., Proc Natl Acad Sci U S A,1999. 96(25): p. 14523-14528).

[0729] Mice bearing established orthotopic LAPC-9 tumors areadministered one to three injections per week of 500-1000 μg of eitheranti-108P5H8 mAb, control antibody or PBS two-to three times per weekover a 4-8 week period. Mice in both groups are allowed to establish ahigh tumor burden (PSA levels greater than 300 ng/ml), to ensure a highfrequency of metastasis formation in mouse lungs. Mice then are killedand their prostate/kidney and lungs are analyzed for the presence oftumor cells by IHC analysis.

[0730] These studies demonstrate a broad anti-tumor efficacy ofanti-108P5H8 antibodies on initiation and/or progression of prostate andkidney cancer in xenograft mouse models. Anti-108P5H8 antibodies inhibittumor formation of both androgen-dependent and androgen-independentprostate tumors as well as retarding the growth of already establishedtumors and prolong the survival of treated mice. Moreover, anti-108P5H8mAbs demonstrate a dramatic inhibitory effect on the spread of localprostate tumor to distal sites, even in the presence of a large tumorburden. Similar therapeutic effects are seen in the kidney cancer model.Thus, anti-108P5H8 mAbs are efficacious-on major clinically relevant endpoints (tumor growth), prolongation of survival, and health.

Example 37

[0731] Therapeutic and Diagnostic Use of Anti-108P5H8 Antibodies inHumans.

[0732] Anti-108P5H8 monoclonal antibodies are safely and effectivelyused for diagnostic, prophylactic, prognostic and/or therapeuticpurposes in humans. Western blot and immunohistochemical analysis ofcancer tissues and cancer xenografts with anti-108P5H8 mAb show strongextensive staining in carcinoma but significantly lower or undetectablelevels in normal tissues. Detection of 108P5H8 in carcinoma and inmetastatic disease demonstrates the usefulness of the mAb as adiagnostic and/or prognostic indicator. Anti-108P5H8 antibodies aretherefore used in diagnostic applications such as immunohistochemistryof biopsy specimens to detect cancer from suspect patients.

[0733] As determined by immunofluorescence, anti-108P5H8 mAbspecifically binds to carcinoma cells. Thus, anti-108P5H8 antibodies areused in diagnostic whole body imaging applications, such asradioimmunoscintigraphy and radioimmunotherapy, (see, e.g., PotamianosS., et. al. Anticancer Res 20(2A):925-948 (2000)) for the detection oflocalized and metastatic cancers that exhibit expression of 108P5H8.Shedding or release of an extracellular domain of 108P5H8 into theextracellular milieu, such as that seen for alkaline phosphodiesteraseBIO (Meerson, N. R., Hepatology 27:563-568 (1998)), allows diagnosticdetection of 108P5H8 by anti-108P5H8 antibodies in serum and/or urinesamples from suspect patients.

[0734] Anti-108P5H8 antibodies that specifically bind 108P5H8 are usedin therapeutic applications for the treatment of cancers that express108P5H8. Anti-108P5H8 antibodies are used as an unconjugated modalityand as conjugated form in which the antibodies are attached to one ofvarious therapeutic or imaging modalities well known in the art, such asa prodrugs, enzymes or radioisotopes. In preclinical studies,unconjugated and conjugated anti-108P5H8 antibodies are tested forefficacy of tumor prevention and growth inhibition in the SCID mousecancer xenograft models, e.g., LAPC9 (see, e.g., the Example entitled“Monoclonal Antibody-mediated Inhibition of Prostate and Kidney TumorsIn vivo.”) Conjugated and unconjugated anti-108P5H8 antibodies are usedas a therapeutic modality in human clinical trials either alone or incombination with other treatments as described in following Examples.

Example 38

[0735] Human Clinical Trials for the Treatment and Diagnosis of HumanCarcinomas Through Use of Human Anti-108P5H8 Antibodies in Vivo

[0736] Antibodies are used in accordance with the present inventionwhich recognize an epitope on 108P5H8, and are used in the treatment ofcertain tumors such as those listed in Table I. Based upon a number offactors, including 108P5H8 expression levels, tumors such as thoselisted in Table I are presently preferred indications. In connectionwith each of these indications, three clinical approaches aresuccessfully pursued.

[0737] I.) Adjunctive therapy: In adjunctive therapy, patients aretreated with anti-108P5H8 antibodies in combination with achemotherapeutic or antineoplastic agent and/or radiation therapy.Primary cancer targets, such as those listed in Table I, are treatedunder standard protocols by the addition anti-108P5H8 antibodies tostandard first and second line therapy. Protocol designs addresseffectiveness as assessed by reduction in tumor mass as well as theability to reduce usual doses of standard chemotherapy. These dosagereductions allow additional and/or prolonged therapy by reducingdose-related toxicity of the chemotherapeutic agent. Anti-108P5H8antibodies are utilized in several adjunctive clinical trials incombination with the chemotherapeutic or antineoplastic agentsadriamycin (advanced prostrate carcinoma), cisplatin (advanced head andneck and lung carcinomas), taxol (breast cancer), and doxorubicin(preclinical).

[0738] II.) Monotherapy: In connection with the use of the anti-108P5H8antibodies in monotherapy of tumors, the antibodies are administered topatients without a chemotherapeutic or antineoplastic agent. In oneembodiment, monotherapy is conducted clinically in end stage cancerpatients with extensive metastatic disease: Patients show some diseasestabilization. Trials demonstrate an effect in refractory patients withcancerous tumors.

[0739] III.) Imaging Agent: Through binding a radionuclide (e.g., iodineor yttrium (I¹³¹, Y⁹⁰) to anti-108P5H8 antibodies, the radiolabeledantibodies are utilized as a diagnostic and/or imaging agent. In such arole, the labeled antibodies localize to both solid tumors, as well as,metastatic lesions of cells expressing 108P5H8. In connection with theuse of the anti-108P5H8 antibodies as imaging agents, the antibodies areused as an adjunct to surgical treatment of solid tumors, as both apre-surgical screen as well as a post-operative follow-up to determinewhat tumor remains and/or returns. In one embodiment, a (¹¹¹In)-108P5H8antibody is used as an imaging agent in a Phase I human clinical trialin patients having a carcinoma that expresses 108P5H8 (by analogy see,e.g., Divgi et al. J. Natl. Cancer Inst. 83:97-104 (1991)). Patients arefollowed with standard anterior and posterior gamma camera. The resultsindicate that primary lesions and metastatic lesions are identified

[0740] Dose and Route of Administration

[0741] As appreciated by those of ordinary skill in the art, dosingconsiderations can be determined through comparison with the analogousproducts that are in the clinic. Thus, anti-108P5H8 antibodies can beadministered with doses in the range of 5 to 400 mg/m², with the lowerdoses used, e.g., in connection with safety studies. The affinity ofanti-108P5H8 antibodies relative to the affinity of a known antibody forits target is one parameter used by those of skill in the art fordetermining analogous dose regimens. Further, anti-108P5H8 antibodiesthat are fully human antibodies, as compared to the chimeric antibody,have slower clearance; accordingly, dosing in patients with such fullyhuman anti-108P5H8 antibodies can be lower, perhaps in the range of 50to 300 mg/m², and still remain efficacious. Dosing in mg/m², as opposedto the conventional measurement of dose in mg/kg, is a measurement basedon surface area and is a convenient dosing measurement that is designedto include patients of all sizes from infants to adults.

[0742] Three distinct delivery approaches are useful for delivery ofanti-108P5H8 antibodies. Conventional intravenous delivery is onestandard delivery technique for many tumors. However, in connection withtumors in the peritoneal cavity, such as tumors of the ovaries, biliaryduct, other ducts, and the like, intraperitoneal administration mayprove favorable for obtaining high dose of antibody at the tumor and toalso minimize antibody clearance. In a similar manner, certain solidtumors possess vasculature that is appropriate for regional perfusion.Regional perfusion allows for a high dose of antibody at the site of atumor and minimizes short term clearance of the antibody.

[0743] Clinical Development Plan (CDP)

[0744] Overview: The CDP follows and develops treatments of anti-108P5H8antibodies in connection with adjunctive therapy, monotherapy, and as animaging agent. Trials initially demonstrate safety and thereafterconfirm efficacy in repeat doses. Trails are open label comparingstandard chemotherapy with standard therapy plus anti-108P5H8antibodies. As will be appreciated, one criteria that can be utilized inconnection with enrollment of patients is 108P5H8 expression levels intheir tumors as determined by biopsy.

[0745] As with any protein or antibody infusion-based therapeutic,safety concerns are related primarily to (i) cytokine release syndrome,i.e., hypotension, fever, shaking, chills; (ii) the development of animmunogenic response to the material (i.e., development of humanantibodies by the patient to the antibody therapeutic, or HAHAresponse); and, (iii) toxicity to normal cells that express 108P5H8.Standard tests and follow-up are utilized to monitor each of thesesafety concerns. Anti-108P5H8 antibodies are found to be safe upon humanadministration.

Example 39

[0746] Human Clinical Trial Adjunctive Therapy with Human Anti-108P5H8Antibody and Chemotherapeutic Agent

[0747] A phase I human clinical trial is initiated to assess the safetyof six intravenous doses of a human anti-108P5H8 antibody in connectionwith the treatment of a solid tumor, e.g., a cancer of a tissue listedin Table I. In the study, the safety of single doses of anti-108P5H8antibodies when utilized as an adjunctive therapy to an antineoplasticor chemotherapeutic agent, such as cisplatin, topotecan, doxorubicin,adriamycin, taxol, or the like, is assessed. The trial design includesdelivery of six single doses of an anti-108P5H8 antibody with dosage ofantibody escalating from approximately about 25 mg/m²to about 275 mg/m²over the course of the treatment in accordance with the followingschedule: Day0 Day7 Day14 Day21 Day28 Day35 mAb Dose 25 75 125 175 225275 mg/m² mg/m² mg/m² mg/m² mg/m² mg/m² Chemotherapy + + + + + +(standard dose)

[0748] Patients are closely followed for one-week following eachadministration of antibody and chemotherapy. In particular, patients areassessed for the safety concerns mentioned above: (i) cytokine releasesyndrome, i.e., hypotension, fever, shaking, chills; (ii) thedevelopment of an immunogenic response to the material (i.e.,development of human antibodies by the patient to the human antibodytherapeutic, or HAHA response); and, (iii) toxicity to normal cells thatexpress 108P5H8. Standard tests and follow-up are utilized to monitoreach of these safety concerns. Patients are also assessed for clinicaloutcome, and particularly reduction in tumor mass as evidenced by MRI orother imaging.

[0749] The anti-108P5H8 antibodies are demonstrated to be safe andefficacious, Phase II trials confirm the efficacy and refine optimumdosing.

Example 40

[0750] Human Clinical Trial: Monotherapy with Human Anti-108P5H8Antibody

[0751] Anti-108P5H8 antibodies are safe in connection with theabove-discussed adjunctive trial, a Phase II human clinical trialconfirms the efficacy and optimum dosing for monotherapy. Such trial isaccomplished, and entails the same safety and outcome analyses, to theabove-described adjunctive trial with the exception being that patientsdo not receive chemotherapy concurrently with the receipt of doses ofanti-108P5H8 antibodies.

Example 41

[0752] Human Clinical Trial: Diagnostic Imaging with Anti-108P5H8Antibody

[0753] Once again, as the adjunctive therapy discussed above is safewithin the safety criteria discussed above, a human clinical trial isconducted concerning the use of anti-108P5H8 antibodies as a diagnosticimaging agent. The protocol is designed in a substantially similarmanner to those described in the art, such as in Divgi et al. J. Natl.Cancer Inst. 83:97-104 (1991). The antibodies are found to be both safeand efficacious when used as a diagnostic modality.

Example 42

[0754] Homology Comparison of 108P5H8 to Known Sequences

[0755] The 108P5H8 protein is a six-transmembrane type 3 cell surfaceprotein, consisting of 429 amino acids (table XXI). The 108P5H8 proteinhas 2 variant forms (FIG. 3), with 108P5H8v.3 differing from 108P5H8 v.1by one amino acid at position 30 (D to E). This alteration in amino acidat position 30 corresponds to a point mutation at nucleic acid position90, making variant 3 a true SNP. Both 108P5H8 variants, 108P5H8 v.1 and108P5H8 v.3 have a calculated molecular weight of 47.5 kDa, and pI of6.11, and contain an ion efflux motif between amino acid 114-417.Proteins carrying the ion efflux motif are found to increase toleranceto divalent metal ions such as cadmium, zinc, and cobalt. These proteinsare thought to be efflux pumps that remove these ions from cells (KunitoT et al, Biosci Biotechnol Biochem 1996, 60:699).

[0756] The 108P5H8 protein variants show homology to human zinctransporter 4 (gi 11432533); with 108P5H8v.1 sharing 100% identity and100% homology with that protein over the entire length of the protein(FIG. 25). 108P5H8v.2 share 100% identity and 100% homology with humanzinc transporter ZNT4-gi 8134840 over the entire protein. As with thetwo 108P5H8 variants, ZNT4 (gi 8134840) and ZNT4-gi 11432533 differ byone amino acid at position 30, showing the same D to E change observedin 108P5H8v.3 and 108P5H8v.1. Based on sequence homology, 108P5H8 isconserved in various, species, showing high homology to Rat ZNT4 (90%identify shown in FIG. 25) and mouse ZNT4 (91% identity with gi8134841).

[0757] Zinc has been shown to play an important role in the physiologyand pathology of prostate epithelial cells. Zinc ions regulate theactivity of chromatin and plasmalemma structures in seminal plasma, andparticipate in spermadhesin function (Holody D and Strzezek J. ActaBiochini Pol 1999, 46:935). In relation to prostate cancer, zinc wasfound to inhibit the activity of aminopeptidase N in prostate cancercells (Ishii K et al, Int J Cancer 2001, 92:49). Efflux of Zn⁺⁺ from theprostate by 108P5H8 or ZNT4 enhances the endogenous activity ofaminopeptidase N, thereby increasing matrix degradation and tissueinvasion by prostate cancer cells. In addition to its role in invasion,ZNT4 regulates apoptosis and proliferation of prostate cells.Accumulation of Zn⁺⁺ within the prostate induces apoptosis of normalepithelial cells (Feng P et al, Mol Urol 2000, 4:31). Enhancedexpression of ZNT4 in prostate cancer cells and the resulting efflux ofZn⁺⁺, allow reduced apoptosis, survival and proliferation of prostatecancer cells. Finally, intracellular Zn⁺⁺ concentrations play a directrole in regulating gene transcription by zinc finger proteins.

[0758] This information indicates that 108P5H8 plays a role in thegrowth of cancer cells, supports cell survival, and regulates genetranscription by regulating events in the nucleus. Accordingly, when108P5H8 functions as a regulator of cell transformation, tumorformation, or as a modulator of transcription involved in activatinggenes associated with inflammation, tumorigenesis or proliferation,108P5H8 is used for therapeutic, diagnostic, prognostic and/orpreventative purposes.

Example 43

[0759] Identification and Confirmation of Signal Transduction Pathways

[0760] Many mammalian proteins have been reported to interact withsignaling molecules and to participate in regulating signaling pathways.(see, e.g., J. Neurochem. 2001; 76:217-223). Using immunoprecipitationand Western blotting techniques, proteins are identified that associatewith 108P5H8 and mediate signaling events. Several pathways known toplay a role in cancer biology can be regulated by 108P5H8, includingphospholipid pathways such as P13K, AKT, etc, adhesion and migrationpathways, including FAK, Rho, Rac-1, etc, as well as mitogenic/survivalcascades such as ERK, p38, etc. (Cell Growth Differ. 2000,11:279; J.Biol. Chem. 1999, 274:801; Oncogene. 2000, 19:3003, J. Cell Biol. 1997,138:913).

[0761] To confirm that 108P5H8 directly or indirectly activates knownsignal transduction pathways in cells, luciferase (luc) basedtranscriptional reporter assays are carried out in cells expressingindividual genes. These transcriptional reporters containconsensus-binding sites for known transcription factors that liedownstream of well-characterized signal transduction pathways. Thereporters and examples of these associated transcription factors, signaltransduction pathways, and activation stimuli are listed below.

[0762] 1. NFkB-luc, NFkB/Rel; Ik-kinase/SAPK; growth/apoptosis/stress

[0763] 2. SRE-luc, SRF/TCF/ELK1; MAPK/SAPK; growth/differentiation

[0764] 3. AP-1-luc, FOS/JUN; MAPK/SAPK/PKC; growth/apoptosis/stress

[0765] 4. ARE-luc, androgen receptor; steroids/MAPK;growth/differentiation/apoptosis

[0766] 5. p53-luc, p53; SAPK; growth/differentiation/apoptosis

[0767] 6. CRE-luc, CREB/ATF2; PKA/p38; growth/apoptosis/stress

[0768] Gene-mediated effects can be assayed in cells showing mRNAexpression. Luciferase reporter plasmids can be introduced bylipid-mediated transfection (TFX-50, Promega). Luciferase activity, anindicator of relative transcriptional activity, is measured byincubation of cell extracts with luciferin substrate and luminescence ofthe reaction is monitored in a luminometer.

[0769] Signaling pathways activated by 108P5H8 are mapped and used forthe identification and validation of therapeutic targets. When 108P5H8is involved in cell signaling, it is used as target for diagnostic,prognostic, preventative and/or therapeutic purposes.

Example 44

[0770] Involvement in Tumor Progression

[0771] The 108P5H8 gene contributes to the growth of cancer cells. Therole of 108P5H8 in tumor growth is confirmed in a variety of primary andtransfected cell lines including prostate cell lines, as well as NIH 3T3cells engineered to stably express 108P5H8. Parental cells lacking108P5H8 and cells expressing 108P5H8 are evaluated for cell growth usinga well-documented proliferation assay (Fraser, S. P., et al., Prostate2000; 44:61, Johnson, D. E., et al., Anticancer Drugs 1996, 7:288).

[0772] To confirm the role of 108P5H8 in the transformation process, itseffect in colony forming assays is investigated. Parental NIH-3T3 cellslacking 108P5H8 are compared to NIH-3T3 cells expressing 108P5H8, usinga soft agar assay under stringent and more permissive conditions (Song,Z., et al., Cancer Res. 2000; 60:6730).

[0773] To confirm the role of 108P5H8 in invasion and metastasis ofcancer cells, a well-established assay is used, e.g., a Transwell InsertSystem assay (Becton Dickinson) (Cancer Res. 1999; 59:6010). Controlcells, including prostate, colon, bladder and kidney cell lines lacking108P5H8 are compared to corresponding cells expressing 108P5H8. Cellsare loaded with the fluorescent dye, calcein, and plated in the top wellof the Transwell insert coated with a basement membrane analog. Invasionis determined by fluorescence of cells in the lower chamber relative tothe fluorescence of the entire cell population.

[0774] 108P5H8 can also play a role in cell cycle and apoptosis.Parental cells and cells expressing 108P5H8 are compared for differencesin cell cycle regulation using a well-established BrdU assay(Abdel-Malek Z A., J Cell Physiol. 1988, 136:247). In short, cells aregrown under both optimal (full serum) and limiting (low serum)conditions, labeled with BrdU and stained with anti-BrdU Ab andpropidium iodide. Cells are analyzed for entry into the G1, S, and G2Mphases of the cell cycle. Alternatively, the effect of stress onapoptosis is evaluated in control parental cells and cells expressing108P5H8, including normal and tumor prostate, colon and lung cells.Engineered and parental cells are treated with various chemotherapeuticagents, such as etoposide, flutamide, etc., and protein synthesisinhibitors, such as cycloheximide. Cells are stained with annexin V-FITCand cell death is measured by FACS analysis. Modulation of cell death by108P5H8 plays a critical role in regulating tumor progression and tumorload.

[0775] When 108P5H8 plays a role in cell growth, transformation,invasion or apoptosis, it is used as a target for diagnostic,prognostic, preventative and/or therapeutic purposes.

Example 45

[0776] Involvement in Angiogenesis

[0777] Angiogenesis or new capillary blood vessel formation is necessaryfor tumor growth (Hanahan, D., Folkman, J., Cell 1996, 86:353; Folkman,J., Endocrinology 1998, 139:441). Several assays have been developed tomeasure angiogenesis in vitro and in vivo, such as the tissue cultureassays endothelial cell tube formation and endothelial cellproliferation. Using these assays as well as in vitroneo-vascularization, the role of 108P5H8 in angiogenesis, enhancement orinhibition, is confirmed.

[0778] For example, endothelial cells engineered to express 108P5H8 areevaluated using tube formation and proliferation-assays. The effect of108P5H8 is also confirmed in animal models in vivo. For example, cellseither expressing or lacking 108P5H8 are implanted subcutaneously inimmunocompromised mice. Endothelial cell migration and angiogenesis areevaluated 5-15 days later using immunohistochemistry techniques. 108P5H8affects angiogenesis, and it is used as a target for diagnostic,prognostic, preventative and/or therapeutic purposes

Example 46

[0779] Regulation of Transcription

[0780] The cellular localization of 108P5H8 (Table XXI) and its abilityto regulate intracellular zinc ion concentrations, 108P5H8 iseffectively used as a modulator of the transcriptional regulation ofeukaryotic genes. Regulation of gene expression is confirmed, e.g., bystudying gene expression in cells expressing or lacking 108P5H8. Forthis purpose, two types of experiments are performed.

[0781] In the first set of experiments, RNA from parental and108P5H8-expressing cells are extracted and hybridized to commerciallyavailable gene arrays (Clontech) (Smid-Koopman, E., et al., Br. J.Cancer, 2000, 83:246). Resting cells as well as cells treated with FBSor androgen are compared. Differentially expressed genes are identifiedin accordance with procedures known in the art. The differentiallyexpressed genes are then mapped to biological pathways (Chen, K., et al.Thyroid 2001, 11:41.).

[0782] In the second set of experiments, specific transcriptionalpathway activation is evaluated using commercially available(Stratagene) luciferase reporter constructs including: NFkB-luc,SRE-luc, ELK1-luc, ARE-luc, p53-luc, and CRE-luc. These transcriptionalreporters contain consensus binding sites for known transcriptionfactors that lie downstream of well-characterized signal transductionpathways, and represent a good tool to ascertain pathway activation andscreen for positive and negative modulators of pathway activation.

[0783] Accordingly, it is found that 108P5H8 plays a role in generegulation, and it is used as a target for diagnostic, prognostic,preventative and/or therapeutic purposes.

Example 47

[0784] Involvement in Cell Adhesion

[0785] Cell adhesion plays a critical role in tissue colonization andmetastasis. 108P5H8 participates in cellular organization, and as aconsequence cell adhesion and motility. To confirm the role of 108P5H8in the regulation of cell adhesion, control cells lacking 108P5H8 arecompared to cells expressing 108P5H8, using techniques previouslydescribed (see, e.g., Haier et al., Br. J. Cancer, 1999, 80:1867; Lehrand Pienta, J. Natl. Cancer Inst. 1998, 90:118). Briefly, in oneembodiment, cells labeled with a fluorescent indicator, such as calcein,are incubated in tissue culture wells coated with media alone or withmatrix proteins. Adherent cells are detected by fluorimetric analysisand percent adhesion is calculated. In another embodiment, cells lackingor expressing 108P5H8 are analyzed for their ability to mediatecell-cell adhesion using similar experimental techniques as describedabove. Both of these experimental systems are used to identify proteins,antibodies and/or small molecules that modulate cell adhesion toextracellular matrix and cell-cell interaction. Cell adhesion plays acritical role in tumor growth, progression, and, colonization, and108P5H8 is involved in these processes. Thus, 108P5H8 serves as adiagnostic, prognostic, preventative and/or therapeutic modality.

Example 48

[0786] Protein-Protein Association

[0787] Several ion transporters have been shown to interact with otherproteins, thereby regulating gene transcription, gene sequence, as wellas cell growth. Using immunoprecipitation techniques as well as twoyeast hybrid systems, proteins are identified that associate with108P5H8. Immunoprecipitates from cells expressing 108P5H8 and cellslacking 108P5H8 are compared for specific protein-protein associations.

[0788] Studies are performed to confirm the extent of association of108P5H8 with effector molecules, such as nuclear proteins, transcriptionfactors, kinases, phsophates, etc. Studies comparing 108P5H8 positiveand 108P5H8 negative cells as well as studies comparingunstimulated/resting cells and cells treated with epithelial cellactivators, such as cytokines, growth factors, androgen andanti-integrin Ab reveal unique interactions.

[0789] In addition, protein-protein interactions are confirmed using twoyeast hybrid methodology (Curr. Opin. Chem. Biol. 1999, 3:64). A vectorcarrying a library of proteins fused to the activation domain of atranscription factor is introduced into yeast expressing a108P5H8-DNA-binding domain fusion protein and a reporter construct.Protein-protein interaction is detected by colorimetric reporteractivity. Specific association with effector molecules and transcriptionfactors directs one of skill to the mode of action of 108P5H8, and thusidentifies therapeutic, prognostic, preventative and/or diagnostictargets for cancer. This and similar assays are also used to identifyand screen for small molecules that interact with 108P5H8.

[0790] Thus it is found that 108P5H8 associates with proteins and smallmolecules. Accordingly, 108P5H8 and these proteins and small moleculesare used for diagnostic, prognostic, preventative and/or therapeuticpurposes.

Example 49

[0791] Ion Flux Activity

[0792] To confirm that 108P5H8 functions as an ion channel, FACSanalysis and electrophysiology techniques are used (Gergely L, Cook L,Agnello V. Clin Diagn Lab Immunol. 1997;4:70; Skryma R, et al. JPhysiol. 2000, 527: 71). Using FACS analysis and commercially availableindicators (Molecular Probes), parental cells and cells expressing genesunder consideration are compared for their ability to transport calcium,and zinc. Prostate, colon, bladder and kidney normal and tumor celllines are used in these studies. For example cells loaded with calciumresponsive indicators such as Fluo4 and Fura red are incubated in thepresence or absence of ions and analyzed by flow cytometry. Informationderived from these experiments provides a mechanism by which cancercells are regulated. This is particularly true in the case of calcium,as calcium channel inhibitors have been reported to induce the death ofcertain cancer cells, including prostate cancer cell lines (Batra S,Popper L D, Hartley-Asp B. Prostate. 1991,19: 299). It is possible todetermine efflux and influx of zinc using fluoZin 1, a fluorescent Zn⁺⁺indicator detected by FACS in a manner similar to Fluo4 above, or using⁶⁵Zn. Prostate, kidney, bladder or colon cells, engineered to express orlack 108P5H8, will be incubated in the presence of ⁶⁵Zn. Cells will beevaluated over time for uptake and efflux of ⁶⁵Zn (Kim A H et al, BrainRes. 2000, 886:99; Grass G et al, J Bacteriol. 2001, 183:4664).

[0793] Using electrophysiology, uninjected oocytes and oocytes injectedwith gene-specific cRNA are compared for ion channel activity.Patch/voltage clamp assays are performed on oocytes in the presence orabsence of selected ions, including calcium, zinc, etc. Ion channelactivators (such as cAMP/GMP, forskolin, TPA, etc) and inhibitors (suchas calcicludine, conotoxin, TEA, tetrodotoxin, etc) are used to evaluatethe function of 108P5H8 as ion channels (Schweitz H. et al. Proc. Natl.Acad. Sci. 1994. 91:878; Skryma R. et al. Prostate. 1997. 33:112).

[0794] Using any of the assays listed above, we can evaluate the effectof antibodies directed against 108P5H8 on ion transport. Similarly,these assays can be used to identify and evaluate small molecule thatmodulate ion and protein transport.

[0795] When 108P5H8 functions as an ion channel, it is used as a targetfor diagnostic, preventative and therapeutic purposes.

Example 50

[0796] Detection of 108P5H8 Protein in LNCaP Cells, a Prostate CancerCell Line by Immunocytochemistry.

[0797] To assess the expression of 108P5H8 protein in a prostate cancercell line, preparations of cytocentrifuged LNCaP cells were stainedusing a rabbit polyclonal antibody to 108P5H8. Preparations of LNCaPcells were made from three differently treated cell populations toassess whether 108P5H8 is androgen regulated. The LNCaP cellpreparations were made from cells grown in medium containing 10% fetalbovine serum; from cells grown for 72 hours in androgen free, serumdepleted medium (by growing in charcoal dextran stripped medium); orfrom previously androgen starved cells which were subsequentlystimulated with 10 mmol mibolerone (a synthetic androgen) for 48 hours.The cells were spun down, washed twice (in buffer), resuspended (inbuffer) and then centrifuged onto slides (1000 rpm for 2 minutes),allowed to dry and then fixed in acetone for 10 minutes. The cells werethen incubated with rabbit polyclonal 108P5H8 for 3 hours (FIGS. 26A-C),or rabbit IgG (FIG. 26D). The slides were washed three times in bufferthen incubated in DAKO EnVision⁺™ peroxidase conjugated goat anti-rabbitsecondary antibody (DAKO Corporation, Carpenteria, Calif.) for 1 hour.The cells were then washed in buffer, developed using the DAB kit (SIGMAChemicals), counterstained using hematoxylin, and analyzed by brightfield microscopy. The results showed strong expression of 108P5H8 in allthree LNCaP cell preparations demonstrating that expression of 108P5H8can be detected in this prostate cancer cell line and is not androgenrelated. This indicates that antibodies to 108P5H8 are useful indetecting non-androgen related cancer of the prostate; the protein is auseful marker.

Example 51

[0798] Detection of 108P5H8 Protein in Prostate Cancer Patient Specimensby Immunohistochemisttry.

[0799] To assess the expression of 108P5H8 protein, prostate cancerspecimens were obtained from prostate cancer patients and stained usinga rabbit polyclonal antibody to 108P5H8. Frozen tissues were then cutinto 4 micron sections and fixed in acetone for 10 minutes. The sectionswere then incubated with rabbit polyclonal 108P5H8 for 3 hours (FIG.27A), or rabbit IgG (FIG. 27B). The slides were washed three times inbuffer then incubated in DAKO EnVision⁺™ peroxidase conjugated goatanti-rabbit secondary antibody (DAKO Corporation, Carpenteria, Calif.)for 1 hour. The sections were then washed in buffer, developed using theDAB kit (SIGMA Chemicals), counterstained using hematoxylin, andanalyzed by bright field microscopy. The results showed strongexpression in the neoplastic glands of the prostate (FIG. 27A). Theseresults further confirm the utility of 108P5H8 as a prostate tumormarker.

[0800] Throughout this application, various website data content,publications, patent applications and patents are referenced. (Websitesare referenced by their Uniform Resource Locator, or URL, addresses onthe World Wide Web.) The disclosures of each of these references arehereby incorporated by reference herein in their entireties.

[0801] The present invention is not to be limited in scope by theembodiments disclosed herein, which are intended as single illustrationsof individual aspects of the invention, and any that are functionallyequivalent are within the scope of the invention. Various modificationsto the models and methods of the invention, in addition to thosedescribed herein, will become apparent to those skilled in the art fromthe foregoing description and teachings, and are similarly intended tofall within the scope of the invention. Such modifications or otherembodiments can be practiced without departing from the true scope andspirit of the invention.

[0802] Table

[0803] Table I: Tissues that Express 108P5H8 When Malignant

[0804] Prostate

[0805] Bladder

[0806] Kidney

[0807] Colon

[0808] Lung

[0809] Ovary

[0810] Breast

[0811] Pancreas

[0812] Uterus

[0813] Stomach

[0814] TABLE II AMINO ACID ABBREVIATIONS SINGLE LETTER THREE LETTER FULLNAME F Phe phenylalanine L Leu leucine S Ser serine Y Tyr tyrosine C Cyscysteine W Trp tryptophan P Pro proline H His histidine Q Gln glutamineR Arg arginine I Ile isoleucine M Met methionine T Thr threonine N Asnasparagine K Lys lysine V Val valine A Ala alanine D Asp aspartic acid EGlu glutamic acid G Gly glycine

[0815] TABLE III AMINO ACID SUBSTITUTION MATRIX Adapted from the GCGSoftware 9.0 BLOSUM62 amino acid substitution matrix (block substitutionmatrix). The higher the value, the more likely a substitution is foundin related, natural proteins. (See URLwww.ikp.unibe.chlmanual/blosum62.html) A C D E F G H I K L M N P Q R S TV W Y . 4 0 -2 -1 -2 0 -2 -1 -1 -1 -1 -2 -1 -1 -1 1 0 0 -3 -2 A 9 3 -4-2 -3 -3 -1 -3 -1 -1 -3 -3 -3 -3 -1 -1 -1 -2 -2 C 6 2 -3 -1 -1 -3 -1 -4-3 1 -1 0 -2 0 -1 -3 -4 -3 D 5 -3 -2 0 -3 1 -3 -2 0 -1 2 0 0 -1 -2 -3 -2E 6 -3 -1 0 -3 0 0 -3 -4 -3 -3 -2 -2 -1 1 3 F 6 -2 -4 -2 -4 -3 0 -2 -2-2 0 -2 -3 -2 -3 G 8 -3 -1 -3 -2 1 -2 0 0 -1 -2 -3 -2 2 H 4 -3 2 1 -3 -3-3 -3 -2 -1 3 -3 -1 I 5 -2 -1 0 -1 1 2 0 -1 -2 -3 -2 K 4 2 -3 -3 -2 -2-2 -1 1 -2 -1 L 5 -2 -2 0 -1 -1 -1 1 -1 -1 M 6 -2 0 0 1 0 -3 -4 -2 N 7-1 -2 -1 -1 -2 -4 -3 P 5 1 0 -1 -2 -2 -1 Q 5 -1 -1 -3 -3 -2 R 4 1 -2 -3-2 S 5 0 -2 -2 T 4 -3 -1 V 11 2 W 7 Y

[0816] TABLE IV (A) POSITION POSITION POSITION 3 (Primary C Terminus 2(Primary Anchor) Anchor) (Primary Anchor) SUPERMOTIFS A1 TI LVMS FWY A2LIVM ATQ IV MATL A3 VSMA TLI RK A24 YF WIVLMT FI YWLM B7 P VILF MWYA B27RHK FYL WMIVA B44 E D FWYLIMVA B58 ATS FWY LIVMA B62 QL IVMP FWYMIVLAMOTIFS A1 TSM Y A1 DE AS Y A2.1 LM VQIAT V LIMAT A3 LMVISATF CGD KYR HFAA11 VTMLISAGN CDF K RYH A24 YFWM FLIW A*3101 MVT ALIS R K A*3301 MVALFIST RK A*6801 AVT MSLI RK B*0702 P LMF WYAIV B*3501 P LMFWY IVA B51 PLIVF WYAM B*5301 P IMFWY ALV B*5401 P ATIV LMFWY

[0817] Bolded residues are preferred, italicized residues are lesspreferred: A peptide is considered motif-bearing if it has primaryanchors at each primary anchor position for a motif or supermotif asspecified in the above table. TABLE IV (B) HLA CLASS II SUPERMOTIF 1 6 9W, F, Y, V, I, L A, V, I, L, P, C, S, T A, V, I, L, C, S, T, M, Y

[0818] TABLE IV (C) MOTIFS 1° anchor 1 2 3 4 5 1° anchor 6 7 8 9 DR4preferred FMYLIVW M T I VSTCPALIM MH MH deleterious W R WDE DR1preferred MFLIVWY PAMQ VMATSPLIC M AVM deleterious C CH FD CWD GDE D DR7preferred MFLIVWY M W A IVMSACTPL M IV deleterious C G GRD N G DR3MOTIFS 1° anchor 1 2 3 1° anchor 4 5 1° anchor 6 motif a LIVMFY Dpreferred motif b LIVMFAY DNQEST KRH preferred DR MFLIVWY VMSTACPLISuper- motif

[0819] TABLE V (A) HLA PEPTIDE SCORING RESULTS - 108P5H8 - A1, 9-MERSSCORE (ESTIMATE OF HALF TIME OF DISASSOCIATION OF A MOLECULE SUBSEQUENCECONTAINING START RESIDUE THIS SEQ. RANK POSITION LISTING SUBSEQUENCE ID#1 23 LNDTSAFDF 6.250 1. 2 148 LTDLSAIIL 6.250 2. 3 141 MTDALHMLT 6.2503. 4 303 IADPICTYV 5.000 4. 5 94 SCDNCSKQR 5.000 5. 6 356 SVEDLNIWS4.500 6. 7 192 YILMGFLLY 2.500 7. 8 377 QLIPGSSSK 2.000 8. 9 178RLEVLSAMI 1.800 9. 10 54 GSEAPERPV 1.350 10. 11 184 AMISVLLVY 1.250 11.12 35 AGDEGLSRF 1.250 12. 13 285 SVGVLIAAY 1.000 13. 14 113 RLTIAAVLY1.000 14. 15 76 LLDQDLPLT 1.000 15. 16 101 QREILKQRK 0.900 16. 17 331ILEGVPSHL 0.900 17. 18 166 KSPTKRFTF 0.750 18. 19 31 FSDEAGDEG 0.750 19.20 214 NGDIMLITA 0.625 20. 21 91 KVDSCDNCS 0.500 21. 22 34 EAGDEGLSR0.500 22. 23 116 IAAVLYLLF 0.500 23. 24 288 VLIAAYIIR 0.500 24. 25 405RCTIQLQSY 0.500 25. 26 340 NVDYIKEAL 0.500 26. 27 226 VAVNVIMGF 0.50027. 28 289 LIAAYIIRF 0.500 28. 29 396 LLLNTFGMY 0.500 29. 30 125MIGELVGGY 0.500 30. 31 397 LLNTFGMYR 0.500 31. 32 302 KIADPICTY 0.50032. 33 83 LTNSQLSLK 0.500 33. 34 189 LLVYILMGF 0.500 34. 35 162WLSSKSPTK 0.400 35. 36 267 DSLAVRAAF 0.300 36. 37 3 GSGAWKRLK 0.300 37.38 70 QADDDSLLD 0.250 38. 39 406 CTIQLQSYR 0.250 39. 40 335 VPSHLNVDY0.250 40. 41 126 IGELVGGYI 0.225 41. 42 290 IAAYIIRFK 0.200 42. 43 310YVFSLLVAF 0.200 43. 44 248 SLPSNSPTR 0.200 44. 45 197 FLLYEAVQR 0.20045. 46 158 LLALWLSSK 0.200 46. 47 98 CSKQREILK 0.150 47. 48 265GQDSLAVRA 0.150 48. 49 247 HSLPSNSPT 0.150 49. 50 414 RQEVDRTCA 0.13550.

[0820] TABLE VI (A) HLA PEPTIDE SCORING RESULTS - 108P5H8 - A1, 10-MERSSCORE (ESTIMATE OF HALF TIME OF DISASSOCIATION OF A MOLECULE SUBSEQUENCECONTAINING START RESIDUE THIS SEQ. RANK POSITION LISTING SUBSEQUENCE ID#1 303 IADPICTYVF 100.000 51. 2 91 KVDSCDNCSK 10.000 52. 3 148 LTDLSAIILT6.250 53. 4 36 GDEGLSRFNK 4.500 54. 5 178 RLEVLSAMIS 4.500 55. 6 247HSLPSNSPTR 3.000 56. 7 54 GSEAPERPVN 2.700 57. 8 76 LLDQDLPLTN 2.500 58.9 183 SAMISVLLVY 2.500 59. 10 70 QADDDSLLDQ 2.500 60. 11 284 QSVGVLIAAY1.500 61. 12 340 NVDYIKEALM 1.000 62. 13 334 GVPSHLNVDY 1.000 63. 14 356SVEDLNIWSL 0.900 64. 15 331 ILEGVPSHLN 0.900 65. 16 31 FSDEAGDEGL 0.75066. 17 141 MTDALHMLTD 0.625 67. 18 396 LLLNTFGMYR 0.500 68. 19 225GVAVNVIMGF 0.500 69. 20 22 FLNDTSAFDF 0.500 70. 21 287 GVLIAAYIIR 0.50071. 22 346 EALMKIEDVY 0.500 72. 23 51 ADDGSEAPER 0.500 73. 24 188VLLVYILMGF 0.500 74. 25 395 HLLLNTFGMY 0.500 75. 26 288 VLIAAYIIRF 0.50076. 27 115 TIAAVLYLLF 0.500 77. 28 126 IGELVGGYIA 0.450 78. 29 165SKSPTKRFTF 0.250 79. 30 202 AVQRTIHMNY 0.250 80. 31 124 FMIGELVGGY 0.25081. 32 279 LGDLVQSVGV 0.250 82. 33 298 KPEYKIADPI 0.225 83. 34 210NYEINGDIML 0.225 84. 35 157 TLLALWLSSK 0.200 85. 36 289 LIAAYIIRFK 0.20086. 37 350 KIEDVYSVED 0.180 87. 38 93 DSCDNCSKQR 0.150 88. 39 312FSLLVAFTTF 0.150 89. 40 414 RQEVDRTCAN 0.135 90. 41 15 RKDDAPLFLN 0.12591. 42 156 LTLLALWLSS 0.125 92. 43 83 LTNSQLSLKV 0.125 93. 44 172FTFGFHRLEV 0.125 94. 45 214 NGDIMLITAA 0.125 95. 46 308 CTYVFSLLVA 0.12596. 47 23 LNDTSAFDFS 0.125 97. 48 35 AGDEGLSRFN 0.125 98. 49 191VYILMGFLLY 0.125 99. 50 97 NCSKQREILK 0.100 100.

[0821] TABLE VII (A) HLA PEPTIDE SCORING RESULTS - 108P5H8 - A2, 9-MERSSCORE (ESTIMATE OF HALF TIME OF DISASSOCIATION OF A MOLECULE SUBSEQUENCECONTAINING START RESIDUE THIS SEQ. RANK POSITION LISTING SUBSEQUENCE ID#1 278 ALGDLVQSV 655.875 101. 2 268 SLAVRAAFV 382.536 102. 3 198LLYEAVQRT 382.282 103. 4 75 SLLDQDLPL 324.068 104. 5 140 IMTDALHML247.333 105. 6 122 LLFMIGELV 214.366 106. 7 155 ILTLLALWL 199.738 107. 8218 MLITAAVGV 118.238 108. 9 121 YLLFMIGEL 108.713 109. 10 323 IIWDTVVII78.258 110. 11 409 QLQSYRQEV 69.552 111. 12 402 GMYRCTIQL 49.371 112. 13118 AVLYLLFMI 45.057 113. 14 181 VLSAMISVL 34.246 114. 15 185 MISVLLVYI30.849 115. 16 147 MLTDLSAII 29.814 116. 17 190 LVYILMGFL 16.722 117. 1869 LQADDDSLL 15.096 118. 19 194 LMGFLLYEA 14.029 119. 20 275 FVHALGDLV13.717 120. 21 308 CTYVFSLLV 11.747 121. 22 76 LLDQDLPLT 11.655 122. 23137 SLAIMTDAL 10.468 123. 24 68 TLQADDDSL 10.468 124. 25 315 LVAFTTFRI9.001 125. 26 183 SAMISVLLV 8.221 126. 27 327 TVVIILEGV 6.859 127. 28115 TIAAVLYLL 6.756 128. 29 153 AIILTLLAL 6.756 129. 30 319 TTFRIIWDT6.606 130. 31 114 LTIAAVLYL 6.381 131. 32 87 QLSLKVDSC 5.599 132. 33 364SLTSGKSTA 4.968 133. 34 357 VEDLNIWSL 4.872 134. 35 312 FSLLVAFTT 4.802135. 36 193 ILMGFLLYE 4.506 136. 37 180 EVLSAMISV 3.884 137. 38 150DLSAIILTL 3.685 138. 39 389 VQSKANHLL 3.682 139. 40 395 HLLLNTFGM 3.625140. 41 303 IADPICTYV 3.613 141. 42 227 AVNVIMGFL 3.074 142. 43 49VVADDGSEA 3.030 143. 44 133 YIANSLAIM 2.963 144. 45 13 MLRKDDAPL 2.760145. 46 216 DIMLITAAV 2.654 146. 47 139 AIMTDALHM 2.527 147. 48 187SVLLVYILM 2.413 148. 49 331 ILEGVPSHL 2.324 149. 50 146 HMLTDLSAI 2.180150.

[0822] TABLE VIII (A) HLA PEPTIDE SCORING RESULTS - 108P5H8 - A2,10-MERS SCORE (ESTIMATE OF HALF TIME OF DISASSOCIATION OF A MOLECULESUBSEQUENCE CONTAINING START RESIDUE THIS SEQ. RANK POSITION LISTINGSUBSEQUENCE ID# 1 302 KIADPICTYV 754.791 151. 2 121 YLLFMIGELV 580.050152. 3 217 IMLITAAVGV 315.959 153. 4 197 FLLYEAVQRT 291.716 154. 5 113RLTIAAVLYL 270.234 155. 6 75 SLLDQDLPLT 260.008 156. 7 323 IIWDTVVIIL160.242 157. 8 310 YVFSLLVAFT 140.388 158. 9 314 LLVAFTTFRI 102.867 159.10 154 IILTLLALWL 101.617 160. 11 184 AMISVLLVYI 95.315 161. 12 330IILEGVPSHL 75.751 162. 13 193 ILMGFLLYEA 71.872 163. 14 39 GLSRFNKLRV69.552 164. 15 194 LMGFLLYEAV 62.765 165. 16 160 ALWLSSKSPT 61.852 166.17 147 MLTDLSAIIL 61.047 167. 18 189 LLVYILMGFL 59.722 168. 19 190LVYILMGFLL 58.977 169. 20 12 SMLRKDDAPL 57.085 170. 21 198 LLYEAVQRTI46.539 171. 22 408 IQLQSYRQEV 44.356 172. 23 140 IMTDALHMLT 37.513 173.24 181 VLSAMISVLL 36.316 174. 25 139 AIMTDALHML 24.997 175. 26 68TLQADDDSLL 21.362 176. 27 397 LLNTFGMYRC 19.425 177. 28 172 FTFGFHRLEV16.441 178. 29 219 LITAAVGVAV 16.258 179. 30 227 AVNVIMGFLL 10.841 180.31 364 SLTSGKSTAI 10.433 181. 32 356 SVEDLNIWSL 8.461 182. 33 318FTTFRIIWDT 8.213 183. 34 22 FLNDTSAFDF 8.152 184. 35 125 MIGELVGGYI7.149 185. 36 348 LMKIEDVYSV 6.874 186. 37 345 KEALMKIEDV 5.335 187. 38179 LEVLSAMISV 5.288 188. 39 285 SVGVLIAAYI 5.021 189. 40 144 ALHMLTDLSA4.968 190. 41 133 YIANSLAIMT 4.713 191. 42 185 MISVLLVYIL 4.709 192. 43150 DLSAIILTLL 3.685 193. 44 389 VQSKANHLLL 3.682 194. 45 129 LVGGYIANSL3.178 195. 46 319 TTFRIIWDTV 2.977 196. 47 307 ICTYVFSLLV 2.933 197. 485 GAWKRLKSML 2.463 198. 49 401 FGMYRCTIQL 2.373 199. 50 332 LEGVPSHLNV2.299 200.

[0823] TABLE IX (A) HLA PEPTIDE SCORING RESULTS - 108P5H8 - A3, 9-MERSSCORE (ESTIMATE OF HALF TIME OF DISASSOCIATION OF A MOLECULE SUBSEQUENCECONTAINING START RESIDUE THIS SEQ. RANK POSITION LISTING SUBSEQUENCE ID#1 158 LLALWLSSK 90.000 201. 2 377 QLIPGSSSK 45.000 202. 3 184 AMISVLLVY27.000 203. 4 397 LLNTFGMYR 24.000 204. 5 162 WLSSKSPTK 20.000 205. 6 39GLSRFNKLR 18.000 206. 7 402 GMYRCTIQL 18.000 207. 8 314 LLVAFTTFR 18.000208. 9 189 LLVYILMGF 13.500 209. 10 288 VLIAAYIIR 12.000 210. 11 361NIWSLTSGK 10.000 211. 12 347 ALMKIEDVY 9.000 212. 13 313 SLLVAFTTF 9.000213. 14 197 FLLYEAVQR 6.000 214. 15 396 LLLNTFGMY 5.400 215. 16 113RLTIAAVLY 4.000 216. 17 248 SLPSNSPTR 4.000 217. 18 192 YILMGFLLY 3.600218. 19 302 KIADPICTY 2.700 219. 20 198 LLYEAVQRT 2.250 220. 21 294IIRFKPEYK 2.000 221. 22 289 LIAAYIIRF 1.800 222. 23 293 YIIRFKPEY 1.800223. 24 234 FLLNQSGHR 1.800 224. 25 75 SLLDQDLPL 1.800 225. 26 122LLFMIGELV 1.500 226. 27 310 YVFSLLVAF 1.500 227. 28 83 LTNSQLSLK 1.500228. 29 146 HMLTDLSAI 1.350 229. 30 331 ILEGVPSHL 1.350 230. 31 155ILTLLALWL 1.200 231. 32 181 VLSAMISVL 0.900 232. 33 395 HLLLNTFGM 0.900233. 34 194 LMGFLLYEA 0.900 234. 35 323 IIWDTVVII 0.900 235. 36 140IMTDALHML 0.900 236. 37 150 DLSAIILTL 0.810 237. 38 287 GVLIAAYII 0.810238. 39 100 KQREILKQR 0.608 239. 40 278 ALGDLVQSV 0.600 240. 41 409QLQSYRQEV 0.600 241. 42 137 SLAIMTDAL 0.600 242. 43 68 TLQADDDSL 0.600243. 44 285 SVGVLIAAY 0.600 244. 45 178 RLEVLSAMI 0.600 245. 46 119VLYLLFMIG 0.600 246. 47 147 MLTDLSAII 0.600 247. 48 13 MLRKDDAPL 0.600248. 49 87 QLSLKVDSC 0.600 249. 50 170 KRFTFGFHR 0.540 250.

[0824] TABLE X (A) HLA PEPTIDE SCORING RESULTS - 108P5H8 - A3, 10-MERSSCORE (ESTIMATE OF HALF TIME OF DISASSOCIATION OF A MOLECULE SUBSEQUENCECONTAINING START RESIDUE THIS SEQ. RANK POSITION LISTING SUBSEQUENCE ID#1 157 TLLALWLSSK 135.000 251. 2 396 LLLNTFGMYR 36.000 252. 3 288VLIAAYIIRF 27.000 253. 4 313 SLLVAFTTFR 18.000 254. 5 188 VLLVYILMGF13.500 255. 6 124 FMIGELVGGY 8.100 256. 7 22 FLNDTSAFDF 6.000 257. 8 91KVDSCDNCSK 6.000 258. 9 104 ILKQRKVKAR 6.000 259. 10 395 HLLLNTFGMY5.400 260. 11 162 WLSSKSPTKR 4.000 261. 12 287 GVLIAAYIIR 3.600 262. 13113 RLTIAAVLYL 3.600 263. 14 293 YIIRFKPEYK 3.000 264. 15 82 PLTNSQLSLK3.000 265. 16 100 KQREILKQRK 2.700 266. 17 323 IIWDTVVIIL 2.700 267. 18314 LLVAFTTFRI 2.700 268. 19 225 GVAVNVIMGF 2.700 269. 20 193 ILMGFLLYEA2.025 270. 21 13 MLRKDDAPLF 2.000 271. 22 348 LMKIEDVYSV 1.800 272. 23184 AMISVLLVYI 1.350 273. 24 334 GVPSHLNVDY 1.200 274. 25 147 MLTDLSAIIL1.200 275. 26 39 GLSRFNKLRV 1.200 276. 27 202 AVQRTIHMNY 1.200 277. 2820 PLFLNDTSAF 1.000 278. 29 376 IQLIPGSSSK 0.900 279. 30 190 LVYILMGFLL0.900 280. 31 181 VLSAMISVLL 0.900 281. 32 12 SMLRKDDAPL 0.900 282. 33119 VLYLLFMIGE 0.900 283. 34 397 LLNTFGMYRC 0.900 284. 35 146 HMLTDLSAII0.900 285. 36 198 LLYEAVQRTI 0.675 286. 37 399 NTFGMYRCTI 0.675 287. 3868 TLQADDDSLL 0.600 288. 39 364 SLTSGKSTAI 0.600 289. 40 268 SLAVRAAFVH0.600 290. 41 270 AVRAAFVHAL 0.540 291. 42 185 MISVLLVYIL 0.540 292. 43160 ALWLSSKSPT 0.500 293. 44 402 GMYRCTIQLQ 0.450 294. 45 289 LIAAYIIRFK0.450 295. 46 338 HLNVDYIKEA 0.450 296. 47 121 YLLFMIGELV 0.450 297. 48197 FLLYEAVQRT 0.450 298. 49 377 QLIPGSSSKW 0.450 299. 50 150 DLSAIILTLL0.405 300.

[0825] TABLE XI (A) HLA PEPTIDE SCORING RESULTS - 108P5H8 - A11, 9-MERSSCORE (ESTIMATE OF HALF TIME OF DISASSOCIATION OF A MOLECULE SUBSEQUENCECONTAINING START RESIDUE THIS SEQ. RANK POSITION LISTING SUBSEQUENCE ID#1 83 LTNSQLSLK 1.000 301. 2 361 NIWSLTSGK 0.800 302. 3 377 QLIPGSSSK0.600 303. 4 294 IIRFKPEYK 0.400 304. 5 162 WLSSKSPTK 0.400 305. 6 158LLALWLSSK 0.400 306. 7 342 DYIKEALMK 0.360 307. 8 406 CTIQLQSYR 0.300308. 9 288 VLIAAYIIR 0.240 309. 10 8 KRLKSMLRK 0.180 310. 11 287GVLIAAYII 0.180 311. 12 100 KQREILKQR 0.180 312. 13 397 LLNTFGMYR 0.160313. 14 39 GLSRFNKLR 0.120 314. 15 197 FLLYEAVQR 0.120 315. 16 234FLLNQSGHR 0.120 316. 17 314 LLVAFTTFR 0.120 317. 18 205 RTIHMNYEI 0.090318. 19 118 AVLYLLFMI 0.090 319. 20 103 EILKQRKVK 0.090 320. 21 248SLPSNSPTR 0.080 321. 22 1 MAGSGAWKR 0.080 322. 23 170 KRFTFGFHR 0.072323. 24 337 SHLNVDYIK 0.060 324. 25 315 LVAFTTFRI 0.060 325. 26 109KVKARLTIA 0.060 326. 27 187 SVLLVYILM 0.060 327. 28 37 DEGLSRFNK 0.054328. 29 402 GMYRCTIQL 0.048 329. 30 190 LVYILMGFL 0.040 330. 31 310YVFSLLVAF 0.040 331. 32 308 CTYVFSLLV 0.040 332. 33 384 SKWEEVQSK 0.040333. 34 98 CSKQREILK 0.040 334. 35 132 GYIANSLAI 0.036 335. 36 327TVVIILEGV 0.030 336. 37 114 LTIAAVLYL 0.030 337. 38 34 EAGDEGLSR 0.024338. 39 275 FVHALGDLV 0.020 339. 40 94 SCDNCSKQR 0.020 340. 41 49VVADDGSEA 0.020 341. 42 282 LVQSVGVLI 0.020 342. 43 290 IAAYIIRFK 0.020343. 44 223 AVGVAVNVI 0.020 344. 45 92 VDSCDNCSK 0.020 345. 46 285SVGVLIAAY 0.020 346. 47 340 NVDYIKEAL 0.020 347. 48 227 AVNVIMGFL 0.020348. 49 270 AVRAAFVHA 0.020 349. 50 101 QREILKQRK 0.020 350.

[0826] TABLE XII (A) HLA PEPTIDE SCORING RESULTS - 108P5H8 - A11,10-MERS SCORE (ESTIMATE OF HALF TIME OF DISASSOCIATION OF A MOLECULESUBSEQUENCE CONTAINING START RESIDUE THIS SEQ. RANK POSITION LISTINGSUBSEQUENCE ID# 1 91 KVDSCDNCSK 6.000 351. 2 287 GVLIAAYIIR 3.600 352. 3100 KQREILKQRK 1.800 353. 4 376 IQLIPGSSSK 0.900 354. 5 157 TLLALWLSSK0.600 355. 6 293 YIIRFKPEYK 0.600 356. 7 97 NCSKQREILK 0.400 357. 8 396LLLNTFGMYR 0.240 358. 9 36 GDEGLSRFNK 0.180 359. 10 196 GFLLYEAVQR 0.180360. 11 233 GFLLNQSGHR 0.180 361. 12 102 REILKQRKVK 0.135 362. 13 225GVAVNVIMGF 0.120 363. 14 190 LVYILMGFLL 0.120 364. 15 109 KVKARLTIAA0.120 365. 16 410 LQSYRQEVDR 0.120 366. 17 405 RCTIQLQSYR 0.120 367. 18313 SLLVAFTTFR 0.120 368. 19 162 WLSSKSPTKR 0.080 369. 20 341 VDYIKEALMK0.080 370. 21 360 LNIWSLTSGK 0.060 371. 22 334 GVPSHLNVDY 0.060 372. 23227 AVNVIMGFLL 0.060 373. 24 253 SPTRGSGCER 0.040 374. 25 7 WKRLKSMLRK0.040 375. 26 104 ILKQRKVKAR 0.040 376. 27 308 CTYVFSLLVA 0.040 377. 28282 LVQSVGVLIA 0.040 378. 29 289 LIAAYIIRFK 0.040 379. 30 172 FTFGFHRLEV0.040 380. 31 356 SVEDLNIWSL 0.040 381. 32 82 PLTNSQLSLK 0.040 382. 33202 AVQRTIHMNY 0.040 383. 34 161 LWLSSKSPTK 0.030 384. 35 48 VVVADDGSEA0.030 385. 36 114 LTIAAVLYLL 0.030 386. 37 353 DVYSVEDLNI 0.024 387. 3839 GLSRFNKLRV 0.024 388. 39 113 RLTIAAVLYL 0.024 389. 40 319 TTFRIIWDTV0.020 390. 41 223 AVGVAVNVIM 0.020 391. 42 383 SSKWEEVQSK 0.020 392. 43270 AVRAAFVHAL 0.020 393. 44 399 NTFGMYRCTI 0.020 394. 45 83 LTNSQLSLKV0.020 395. 46 285 SVGVLIAAYI 0.020 396. 47 129 LVGGYIANSL 0.020 397. 48340 NVDYIKEALM 0.020 398. 49 314 LLVAFTTFRI 0.018 399. 50 322 RIIWDTVVII0.018 400.

[0827] TABLE XIII (A) HLA PEPTIDE SCORING RESULTS - 108P5H8 - A24,9-MERS SCORE (ESTIMATE OF HALF TIME OF DISASSOCIATION OF A MOLECULESUBSEQUENCE CONTAINING START RESIDUE THIS SEQ. RANK POSITION LISTINGSUBSEQUENCE ID# 1 191 VYILMGFLL 300.000 401. 2 199 LYEAVQRTI 105.000402. 3 132 GYIANSLAI 75.000 403. 4 354 VYSVEDLNI 50.000 404. 5 171RFTFGFHRL 48.000 405. 6 210 NYEINGDIM 37.500 406. 7 274 AFVHALGDL 30.000407. 8 21 LFLNDTSAF 15.000 408. 9 106 KQRKVKARL 11.200 409. 10 38EGLSRFNKL 9.504 410. 11 331 ILEGVPSHL 8.400 411. 12 227 AVNVIMGFL 8.400412. 13 309 TYVFSLLVA 7.500 413. 14 75 SLLDQDLPL 7.200 414. 15 186ISVLLVYIL 7.200 415. 16 388 EVQSKANHL 7.200 416. 17 130 VGGYIANSL 6.720417. 18 307 ICTYVFSLL 6.720 418. 19 121 YLLFMIGEL 6.600 419. 20 153AIILTLLAL 6.000 420. 21 166 KSPTKRFTF 6.000 421. 22 281 DLVQSVGVL 6.000422. 23 68 TLQADDDSL 6.000 423. 24 143 DALHMLTDL 6.000 424. 25 228VNVIMGFLL 6.000 425. 26 80 DLPLTNSQL 6.000 426. 27 114 LTIAAVLYL 6.000427. 28 150 DLSAIILTL 5.600 428. 29 340 NVDYIKEAL 5.600 429. 30 324IWDTVVIIL 5.600 430. 31 115 TIAAVLYLL 5.600 431. 32 370 STAIVHIQL 5.600432. 33 151 LSAIILTLL 5.600 433. 34 182 LSAMISVLL 5.600 434. 35 300EYKIADPIC 5.000 435. 36 400 TFGMYRCTI 5.000 436. 37 412 SYRQEVDRT 5.000437. 38 6 AWKRLKSML 4.800 438. 39 190 LVYILMGFL 4.800 439. 40 69LQADDDSLL 4.800 440. 41 155 ILTLLALWL 4.800 441. 42 140 IMTDALHML 4.800442. 43 189 LLVYILMGF 4.200 443. 44 226 VAVNVIMGF 4.200 444. 45 267DSLAVRAAF 4.200 445. 46 13 MLRKDDAPL 4.000 446. 47 402 GMYRCTIQL 4.000447. 48 2 AGSGAWKRL 4.000 448. 49 97 NCSKQREIL 4.000 449. 50 390QSKANHLLL 4.000 450.

[0828] TABLE XIV (A) HLA PEPTIDE SCORING RESULTS - 108P5H8 - A24,10-MERS SCORE (ESTIMATE OF HALF TIME OF DISASSOCIATION OF A MOLECULESUBSEQUENCE CONTAINING START RESIDUE THIS SEQ. RANK POSITION LISTINGSUBSEQUENCE ID# 1 120 LYLLFMIGEL 330.000 451. 2 210 NYEINGDIML 300.000452. 3 309 TYVFSLLVAF 180.000 453. 4 342 DYIKEALMKI 82.500 454. 5 132GYIANSLAIM 37.500 455. 6 173 TFGFHRLEVL 20.000 456. 7 60 RPVNGAHPTL12.000 457. 8 369 KSTAIVHIQL 11.200 458. 9 111 KARLTIAAVL 11.200 459. 10339 LNVDYIKEAL 10.080 460. 11 330 IILEGVPSHL 10.080 461. 12 191VYILMGFLLY 9.000 462. 13 392 KANHLLLNTF 8.640 463. 14 226 VAVNVIMGFL8.400 464. 15 114 LTIAAVLYLL 8.400 465. 16 292 AYIIRFKPEY 8.250 466. 17113 RLTIAAVLYL 8.000 467. 18 189 LLVYILMGFL 7.200 468. 19 356 SVEDLNIWSL7.200 469. 20 154 IILTLLALWL 7.200 470. 21 403 MYRCTIQLQS 7.000 471. 22412 SYRQEVDRTC 7.000 472. 23 129 LVGGYIANSL 6.720 473. 24 323 IIWDTVVIIL6.720 474. 25 305 DPICTYVFSL 6.000 475. 26 152 SAIILTLLAL 6.000 476. 27401 FGMYRCTIQL 6.000 477. 28 12 SMLRKDDAPL 6.000 478. 29 68 TLQADDDSLL6.000 479. 30 354 VYSVEDLNIW 6.000 480. 31 81 LPLTNSQLSL 6.000 481. 32227 AVNVIMGFLL 6.000 482. 33 139 AIMTDALHML 6.000 483. 34 136 NSLAIMTDAL6.000 484. 35 388 EVQSKANHLL 6.000 485. 36 180 EVLSAMISVL 6.000 486. 3774 DSLLDQDLPL 6.000 487. 38 150 DLSAIILTLL 5.600 488. 39 181 VLSAMISVLL5.600 489. 40 300 EYKIADPICT 5.000 490. 41 185 MISVLLVYIL 4.800 491. 425 GAWKRLKSML 4.800 492. 43 147 MLTDLSAIIL 4.800 493. 44 31 FSDEAGDEGL4.800 494. 45 298 KPEYKIADPI 4.200 495. 46 188 VLLVYILMGF 4.200 496. 47273 AAFVHALGDL 4.000 497. 48 270 AVRAAFVHAL 4.000 498. 49 1 MAGSGAWKRL4.000 499. 50 96 DNCSKQREIL 4.000 500.

[0829] TABLE XV (A) HLA PEPTIDE SCORING RESULTS - 108P5H8 - B7, 9-MERSSCORE (ESTIMATE OF HALF TIME OF DISASSOCIATION OF A MOLECULE SUBSEQUENCECONTAINING START RESIDUE THIS SEQ. RANK POSITION LISTING SUBSEQUENCE ID#1 227 AVNVIMGFL 60.000 501. 2 106 KQRKVKARL 40.000 502. 3 13 MLRKDDAPL40.000 503. 4 190 LVYILMGFL 20.000 504. 5 388 EVQSKANHL 20.000 505. 6270 AVRAAFVHA 15.000 506. 7 2 AGSGAWKRL 12.000 507. 8 143 DALHMLTDL12.000 508. 9 153 AIILTLLAL 12.000 509. 10 139 AIMTDALHM 9.000 510. 11117 AAVLYLLFM 9.000 511. 12 340 NVDYIKEAL 6.000 512. 13 97 NCSKQREIL6.000 513. 14 111 KARLTIAAV 6.000 514. 15 118 AVLYLLFMI 6.000 515. 16223 AVGVAVNVI 6.000 516. 17 187 SVLLVYILM 5.000 517. 18 150 DLSAIILTL4.000 518. 19 307 ICTYVFSLL 4.000 519. 20 186 ISVLLVYIL 4.000 520. 21130 VGGYIANSL 4.000 521. 22 151 LSAIILTLL 4.000 522. 23 182 LSAMISVLL4.000 523. 24 390 QSKANHLLL 4.000 524. 25 155 ILTLLALWL 4.000 525. 26402 GMYRCTIQL 4.000 526. 27 75 SLLDQDLPL 4.000 527. 28 69 LQADDDSLL4.000 528. 29 181 VLSAMISVL 4.000 529. 30 370 STAIVHIQL 4.000 530. 31114 LTIAAVLYL 4.000 531. 32 281 DLVQSVGVL 4.000 532. 33 80 DLPLTNSQL4.000 533. 34 389 VQSKANHLL 4.000 534. 35 38 EGLSRFNKL 4.000 535. 36 115TIAAVLYLL 4.000 536. 37 140 IMTDALHML 4.000 537. 38 68 TLQADDDSL 4.000538. 39 174 FGFHRLEVL 4.000 539. 40 228 VNVIMGFLL 4.000 540. 41 121YLLFMIGEL 4.000 541. 42 137 SLAIMTDAL 4.000 542. 43 5 GAWKRLKSM 3.000543. 44 201 EAVQRTIHM 3.000 544. 45 57 APERPVNGA 2.700 545. 46 61PVNGAHPTL 2.000 546. 47 282 LVQSVGVLI 2.000 547. 48 287 GVLIAAYII 2.000548. 49 315 LVAFTTFRI 2.000 549. 50 40 LSRFNKLRV 2.000 550.

[0830] TABLE XVI (A) HLA PEPTIDE SCORING RESULTS - 108P5H8 - B7, 10-MERSSCORE (ESTIMATE OF HALF TIME OF DISASSOCIATION OF A MOLECULE SUBSEQUENCECONTAINING START RESIDUE THIS SEQ. RANK POSITION LISTING SUBSEQUENCE ID#1 270 AVRAAFVHAL 600.000 551. 2 111 KARLTIAAVL 120.000 552. 3 60RPVNGAHPTL 80.000 553. 4 81 LPLTNSQLSL 80.000 554. 5 305 DPICTYVFSL80.000 555. 6 227 AVNVIMGFLL 60.000 556. 7 273 AAFVHALGDL 36.000 557. 8139 AIMTDALHML 36.000 558. 9 388 EVQSKANHLL 20.000 559. 10 180EVLSAMISVL 20.000 560. 11 190 LVYILMGFLL 20.000 561. 12 129 LVGGYIANSL20.000 562. 13 223 AVGVAVNVIM 15.000 563. 14 5 GAWKRLKSML 12.000 564. 151 MAGSGAWKRL 12.000 565. 16 152 SAIILTLLAL 12.000 566. 17 401 FGMYRCTIQL12.000 567. 18 226 VAVNVIMGFL 12.000 568. 19 335 VPSHLNVDYI 8.000 569.20 356 SVEDLNIWSL 6.000 570. 21 19 APLFLNDTSA 6.000 571. 22 96DNCSKQREIL 6.000 572. 23 294 IIRFKPEYKI 6.000 573. 24 113 RLTIAAVLYL4.000 574. 25 150 DLSAIILTLL 4.000 575. 26 339 LNVDYIKEAL 4.000 576. 27240 GHRHSHSHSL 4.000 577. 28 147 MLTDLSAIIL 4.000 578. 29 330 IILEGVPSHL4.000 579. 30 114 LTIAAVLYLL 4.000 580. 31 189 LLVYILMGFL 4.000 581. 3212 SMLRKDDAPL 4.000 582. 33 74 DSLLDQDLPL 4.000 583. 34 185 MISVLLVYIL4.000 584. 35 260 CERNHGQDSL 4.000 585. 36 181 VLSAMISVLL 4.000 586. 37323 IIWDTVVIIL 4.000 587. 38 68 TLQADDDSLL 4.000 588. 39 136 NSLAIMTDAL4.000 589. 40 154 IILTLLALWL 4.000 590. 41 369 KSTAIVHIQL 4.000 591. 42389 VQSKANHLLL 4.000 592. 43 117 AAVLYLLFMI 3.600 593. 44 222 AAVGVAVNVI3.600 594. 45 138 LAIMTDALHM 3.000 595. 46 116 IAAVLYLLFM 3.000 596. 47298 KPEYKIADPI 2.400 597. 48 315 LVAFTTFRII 2.000 598. 49 285 SVGVLIAAYI2.000 599. 50 40 LSRFNKLRVV 2.000 600.

[0831] TABLE XVII (A) HLA PEPTIDE SCORING RESULTS - 108P5H8 - B35,9-MERS SCORE (ESTIMATE OF HALF TIME OF DISASSOCIATION OF A MOLECULESUBSEQUENCE CONTAINING START RESIDUE THIS SEQ. RANK POSITION LISTINGSUBSEQUENCE ID# 1 335 VPSHLNVDY 40.000 601. 2 390 QSKANHLLL 15.000 602.3 164 SSKSPTKRF 15.000 603. 4 166 KSPTKRFTF 10.000 604. 5 302 KIADPICTY8.000 605. 6 355 YSVEDLNIW 7.500 606. 7 201 EAVQRTIHM 6.000 607. 8 5GAWKRLKSM 6.000 608. 9 203 VQRTIHMNY 6.000 609. 10 117 AAVLYLLFM 6.000610. 11 106 KQRKVKARL 6.000 611. 12 267 DSLAVRAAF 5.000 612. 13 151LSAIILTLL 5.000 613. 14 182 LSAMISVLL 5.000 614. 15 186 ISVLLVYIL 5.000615. 16 13 MLRKDDAPL 4.500 616. 17 60 RPVNGAHPT 4.000 617. 18 125MIGELVGGY 4.000 618. 19 113 RLTIAAVLY 4.000 619. 20 405 RCTIQLQSY 4.000620. 21 111 KARLTIAAV 3.600 621. 22 143 DALHMLTDL 3.000 622. 23 69LQADDDSLL 3.000 623. 24 116 IAAVLYLLF 3.000 624. 25 40 LSRFNKLRV 3.000625. 26 226 VAVNVIMGF 3.000 626. 27 139 AIMTDALHM 3.000 627. 28 343YIKEALMKI 2.400 628. 29 383 SSKWEEVQS 2.250 629. 30 19 APLFLNDTS 2.000630. 31 192 YILMGFLLY 2.000 631. 32 293 YIIRFKPEY 2.000 632. 33 184AMISVLLVY 2.000 633. 34 75 SLLDQDLPL 2.000 634. 35 187 SVLLVYILM 2.000635. 36 224 VGVAVNVIM 2.000 636. 37 81 LPLTNSQLS 2.000 637. 38 133YIANSLAIM 2.000 638. 39 347 ALMKIEDVY 2.000 639. 40 305 DPICTYVFS 2.000640. 41 285 SVGVLIAAY 2.000 641. 42 140 IMTDALHML 2.000 642. 43 395HLLLNTFGM 2.000 643. 44 396 LLLNTFGMY 2.000 644. 45 68 TLQADDDSL 1.500645. 46 371 TAIVHIQLI 1.200 646. 47 316 VAFTTFRII 1.200 647. 48 322RIIWDTVVI 1.200 648. 49 97 NCSKQREIL 1.000 649. 50 181 VLSAMISVL 1.000650.

[0832] TABLE XVIII (A) HLA PEPTIDE SCORING RESULTS - 108P5H8 - B35,10-MERS SCORE (ESTIMATE OF HALF TIME OF DISASSOCIATION OF A MOLECULESUBSEQUENCE CONTAINING START RESIDUE THIS SEQ. RANK POSITION LISTINGSUBSEQUENCE ID# 1 60 RPVNGAHPTL 40.000 651. 2 305 DPICTYVFSL 20.000 652.3 81 LPLTNSQLSL 20.000 653. 4 167 SPTKRFTFGF 20.000 654. 5 111KARLTIAAVL 18.000 655. 6 369 KSTAIVHIQL 10.000 656. 7 284 QSVGVLIAAY10.000 657. 8 186 ISVLLVYILM 10.000 658. 9 138 LAIMTDALHM 9.000 659. 10335 VPSHLNVDYI 8.000 660. 11 392 KANHLLLNTF 6.000 661. 12 34 EAGDEGLSRF6.000 662. 13 346 EALMKIEDVY 6.000 663. 14 116 IAAVLYLLFM 6.000 664. 15183 SAMISVLLVY 6.000 665. 16 136 NSLAIMTDAL 5.000 666. 17 74 DSLLDQDLPL5.000 667. 18 312 FSLLVAFTTF 5.000 668. 19 163 LSSKSPTKRF 5.000 669. 20298 KPEYKIADPI 4.800 670. 21 13 MLRKDDAPLF 4.500 671. 22 209 MNYEINGDIM4.000 672. 23 40 LSRFNKLRVV 3.000 673. 24 270 AVRAAFVHAL 3.000 674. 25273 AAFVHALGDL 3.000 675. 26 226 VAVNVIMGFL 3.000 676. 27 5 GAWKRLKSML3.000 677. 28 1 MAGSGAWKRL 3.000 678. 29 152 SAIILTLLAL 3.000 679. 30 31FSDEAGDEGL 3.000 680. 31 19 APLFLNDTSA 2.000 681. 32 4 SGAWKRLKSM 2.000682. 33 113 RLTIAAVLYL 2.000 683. 34 395 HLLLNTFGMY 2.000 684. 35 147MLTDLSAIIL 2.000 685. 36 334 GVPSHLNVDY 2.000 686. 37 66 HPTLQADDDS2.000 687. 38 339 LNVDYIKEAL 2.000 688. 39 249 LPSNSPTRGS 2.000 689. 40323 IIWDTVVIIL 2.000 690. 41 330 IILEGVPSHL 2.000 691. 42 202 AVQRTIHMNY2.000 692. 43 124 FMIGELVGGY 2.000 693. 44 22 FLNDTSAFDF 2.000 694. 45223 AVGVAVNVIM 2.000 695. 46 164 SSKSPTKRFT 1.500 696. 47 390 QSKANHLLLN1.500 697. 48 12 SMLRKDDAPL 1.500 698. 49 68 TLQADDDSLL 1.500 699. 50316 VAFTTFRIIW 1.500 700.

[0833] TABLE V (B) unique to variant 3 relative to variants 1 and 2 HLAPEPTIDE SCORING RESULTS - 108P5H8 - A1, 9-MERS SCORE (ESTIMATE OF HALFTIME OF DISASSOCIATION OF A MOLECULE SUBSEQUENCE CONTAINING STARTRESIDUE THIS SEQ. RANK POSITION LISTING SUBSEQUENCE ID# 1 23 LNDTSAFEF6.250 701. 2 28 AFEFSDEAG 0.045 702. 3 25 DTSAFEFSD 0.013 703. 4 28SAFEFSDEA 0.010 704. 5 22 FLNDTSAFE 0.002 705. 6 26 TSAFEFSDE 0.002 706.7 24 NDTSAFEFS 0.001 707. 8 29 FEFSDEAGD 0.000 708. 9 30 EFSDEAGDE 0.000709.

[0834] TABLE VI (B) unique to variant 3 relative to variants 1 and 2 HLAPEPTIDE SCORING RESULTS - 108P5H8 - A1, 10-MERS SCORE (ESTIMATE OF HALFTIME OF DISASSOCIATION OF A MOLECULE SUBSEQUENCE CONTAINING STARTRESIDUE THIS SEQ. RANK POSITION LISTING SUBSEQUENCE ID# 1 22 FLNDTSAFEF0.500 710. 2 23 LNDTSAFEFS 0.125 711. 3 26 TSAFEFSDEA 0.015 712. 4 27SAFEFSDEAG 0.010 713. 5 28 AFEFSDEAGD 0.009 714. 6 25 DTSAFEFSDE 0.003715. 7 30 EFSDEAGDEG 0.001 716. 8 24 NDTSAFEFSD 0.000 717. 9 21LFLNDTSAFE 0.000 718. 10 29 FEFSDEAGDE 0.000 719.

[0835] TABLE VII (B) unique to variant 3 relative to variants 1 and 2HLA PEPTIDE SCORING RESULTS - 108P5H8 - A2, 9-MERS SCORE (ESTIMATE OFHALF TIME OF DISASSOCIATION OF A MOLECULE SUBSEQUENCE CONTAINING STARTRESIDUE THIS SEQ. RANK POSITION LISTING SUBSEQUENCE ID# 1 27 SAFEFSDEA1.949 720. 2 22 FLNDTSAFE 1.546 721. 3 29 FEFSDEAGD 0.005 722. 4 23LNDTSAFEF 0.002 723. 5 24 NDTSAFEFS 0.001 724. 6 25 DTSAFEFSD 0.000 725.7 26 TSAFEFSDE 0.000 726. 8 28 AFEFSDEAG 0.000 727. 9 30 EFSDEAGDE 0.000728.

[0836] TABLE VIII (B) unique to variant 3 relative to variants 1 and 2HLA PEPTIDE SCORING RESULTS - 108P5H8 - A2, 10-MERS SCORE (ESTIMATE OFHALF TIME OF DISASSOCIATION SUBSEQUENCE OF A MOLECULE START RESIDUECONTAINING THIS SEQ. RANK POSITION LISTING SEQUENCE) ID # 1 22FLNDTSAFEF 8.152 729. 2 26 TSAFEFSDEA 0.060 730. 3 27 SAFEFSDEAG 0.008731. 4 23 LNDTSAFEFS 0.002 732. 5 29 FEFSDEAGDE 0.001 733. 6 24NDTSAFEFSD 0.000 734. 7 21 LFLNDTSAFE 0.000 735. 8 25 DTSAFEFSDE 0.000736. 9 30 EFSDEAGDEG 0.000 737. 10 28 AFEFSDEAGD 0.000 738.

[0837] TABLE IX (B) unique to variant 3 relative to variants 1 and 2 HLAPEPTIDE SCORING RESULTS - 108P5H8 - A3, 9-MERS SCORE (ESTIMATE OF HALFTIME OF DISASSOCIATION SUBSEQUENCE OF A MOLECULE START RESIDUECONTAINING THIS SEQ. RANK POSITION LISTING SUBSEQUENCE) ID # 1 27SAFEFSDEA 0.045 739. 2 22 FLNDTSAFE 0.020 740. 3 23 LNDTSAFEF 0.012 741.4 25 DTSAFEFSD 0.003 742. 5 29 FEFSDEAGD 0.000 743. 6 26 TSAFEFSDE 0.000744. 7 24 NDTSAFEFS 0.000 745. 8 28 AFEFSDEAG 0.000 746. 9 30 EFSDEAGDE0.000 747.

[0838] TABLE X (B) unique to variant 3 relative to variants 1 and 2 HLAPEPTIDE SCORING RESULTS - 108P5H8 - A3, 10-MERS SCORE (ESTIMATE OF HALFTIME OF DISASSOCIATION SUBSEQUENCE OF A MOLECULE START RESIDUECONTAINING THIS SEQ. RANK POSITION LISTING SUBSEQUENCE) ID # 1 22FLNDTSAFEF 6.000 748. 2 26 TSAFEFSDEA 0.003 749. 3 27 SAFEFSDEAG 0.002750. 4 25 DTSAFEFSDE 0.001 751. 5 23 LNDTSAFEFS 0.000 752. 6 24NDTSAFEFSD 0.000 753. 7 29 FEFSDEAGDE 0.000 754. 8 21 LFLNDTSAFE 0.000755. 9 28 AFEFSDEAGD 0.000 756. 10 30 EFSDEAGDEG 0.000 757.

[0839] TABLE XI (B) unique to variant 3 relative to variants 1 and 2 HLAPEPTIDE SCORING RESULTS - 108P5H8 - All, 9-MERS SCORE (ESTIMATE OF HALFTIME OF DISASSOCIATION SUBSEQUENCE OF A MOLECULE START RESIDUECONTAINING THIS SEQ. RANK POSITION LISTING SUBSEQUENCE) ID # 1 27SAFEFSDEA 0.004 758. 2 23 LNDTSAFEF 0.001 759. 3 25 DTSAFEFSD 0.001 760.4 22 FLNDTSAFE 0.000 761. 5 28 AFEFSDEAG 0.000 762. 6 29 FEFSDEAGD 0.000763. 7 30 EFSDEAGDE 0.000 764. 8 24 NDTSAFEFS 0.000 765. 9 26 TSAFEFSDE0.000 766.

[0840] TABLE XII (B) unique to variant 3 relative to variants 1 and 2HLA PEPTIDE SCORING RESULTS - 108P5H8 - All, 10-MERS SCORE (ESTIMATE OFHALF TIME OF DISASSOCIATION SUBSEQUENCE OF A MOLECULE START RESIDUECONTAINING THIS SEQ. RANK POSITION LISTING SUBSEQUENCE) ID # 1 22FLNDTSAFEF 0.012 767. 2 27 SAFEFSDEAG 0.000 768. 3 21 LFLNDTSAFE 0.000769. 4 25 DTSAFEFSDE 0.000 770. 5 28 AFEFSDEAGD 0.000 771. 6 26TSAFEFSDEA 0.000 772. 7 29 FEFSDEAGDE 0.000 773. 8 24 NDTSAFEFSD 0.000774. 9 30 EFSDEAGDEG 0.000 775. 10 23 LNDTSAFEFS 0.000 776.

[0841] TABLE XIII (B) unique to variant 3 relative to variants 1 and 2HLA PEPTIDE SCORING RESULTS - 108P5H8 - A24, 9-MERS SCORE (ESTIMATE OFHALF TIME OF DISASSOCIATION SUBSEQUENCE OF A MOLECULE START RESIDUECONTAINING THIS SEQ. RANK POSITION LISTING SUBSEQUENCE) ID # 1 23LNDTSAFEF 2.200 777. 2 27 SAFEFSDEA 0.132 778. 3 28 AFEFSDEAG 0.075 779.4 30 EFSDEAGDE 0.060 780. 5 22 FLNDTSAFE 0.018 781. 6 24 NDTSAFEFS 0.012782. 7 26 TSAFEFSDE 0.012 783. 8 25 DTSAFEFSD 0.010 784. 9 29 FEFSDEAGD0.001 785.

[0842] TABLE XIV (B) unique to variant 3 relative to variants 1 and 2HLA PEPTIDE SCORING RESULTS - 108P5H8 - A24, 10-MERS SCORE (ESTIMATE OFHALF TIME OF DISASSOCIATION SUBSEQUENCE OF A MOLECULE START RESIDUECONTAINING THIS SEQ. RANK POSITION LISTING SUBSEQUENCE) ID # 1 22FLNDTSAFEF 3.960 786. 2 23 LNDTSAFEFS 0.120 787. 3 26 TSAFEFSDEA 0.110788. 4 28 AFEFSDEAGD 0.075 789. 5 21 LFLNDTSAFE 0.075 790. 6 30EFSDEAGDEG 0.066 791. 7 27 SAFEFSDEAG 0.012 792. 8 25 DTSAFEFSDE 0.012793. 9 24 NDTSAFEFSD 0.001 794. 10 29 FEFSDEAGDE 0.001 795.

[0843] TABLE XV (B) unique to variant 3 relative to variants 1 and 2 HLAPEPTIDE SCORING RESULTS - 108P5H8 - B7, 9-MERS SCORE (ESTIMATE OF HALFTIME OF DISASSOCIATION SUBSEQUENCE OF A MOLECULE START RESIDUECONTAINING THIS SEQ. RANK POSITION LISTING SUBSEQUENCE) ID # 1 27SAFEFSDEA 0.300 796. 2 22 FLNDTSAFE 0.010 797. 3 26 TSAFEFSDE 0.010 798.4 25 DTSAFEFSD 0.010 799. 5 23 LNDTSAFEF 0.006 800. 6 24 NDTSAFEFS 0.002801. 7 30 EFSDEAGDE 0.001 802. 8 29 FEFSDEAGD 0.001 803. 9 28 AFEFSDEAG0.001 804.

[0844] TABLE XVI (B) unique to variant 3 relative to variants 1 and 2HLA PEPTIDE SCORING RESULTS - 108P5H8 - B7, 10-MERS SCORE (ESTIMATE OFHALF TIME OF DISASSOCIATION SUBSEQUENCE OF A MOLECULE START RESIDUECONTAINING THIS SEQ. RANK POSITION LISTING SUBSEQUENCE) ID # 1 26TSAFEFSDEA 0.100 805. 2 27 SAFEFSDEAG 0.030 806. 3 22 FLNDTSAFEF 0.020807. 4 25 DTSAFEFSDE 0.010 808. 5 23 LNDTSAFEFS 0.006 809. 6 30EFSDEAGDEG 0.001 810 7 24 NDTSAFEFSD 0.001 811. 8 29 FEFSDEAGDE 0.001812. 9 21 LFLNDTSAFE 0.001 813. 10 28 AFEFSDEAGD 0.001 814.

[0845] TABLE XVII (B) unique to variant 3 relative to variants 1 and 2HLA PEPTIDE SCORING RESULTS - 108P5H8 - B35, 9-MERS SCORE (ESTIMATE OFHALF TIME OF DISASSOCIATION SUBSEQUENCE OF A MOLECULE START RESIDUECONTAINING THIS SEQ. RANK POSITION LISTING SUBSEQUENCE) ID # 1 276SAFEFSDEA 0.600 815. 2 23 LNDTSAFEF 0.300 816. 3 26 TSAFEFSDE 0.075 817.4 22 FLNDTSAFE 0.020 818. 5 24 NDTSAFEFS 0.010 819. 6 25 DTSAFEFSD 0.010820. 7 30 EFSDEAGDE 0.003 821. 8 29 FEFSDEAGD 0.002 822. 9 28 AFEFSDEAG0.000 823.

[0846] TABLE XVIII (B) unique to variant 3 relative to variants 1 and 2HLA PEPTIDE SCORING RESULTS - 108P5H8 - B35, 10-MERS SCORE (ESTIMATE OFHALF TIME OF DISASSOCIATION OF A MOLECULE SUBSEQUENCE CONTAINING STARTRESIDUE THIS SEQ. RANK POSITION LISTING SUBSEQUENCE ID# 1 22 FLNDTSAFEF2.000 824. 2 26 TSAFEFSDEA 0.500 825. 3 27 SAFEFSDEAG 0.060 826. 4 23LNDTSAFEFS 0.030 827. 5 25 DTSAFEFSDE 0.015 828. 6 30 EFSDEAGDEG 0.002829. 7 29 FEFSDEAGDE 0.002 830. 8 24 NDTSAFEFSD 0.001 831. 9 21LFLNDTSAFE 0.001 832. 10 28 AFEFSDEAGD 0.000 833.

[0847] TABLE XIX Motifs and Post-translational Modifications of 108P5H8N-glycosylation site Number of matches: 3 1 24-27 NDTS (SEQ. ID. No.834) 2  97-100 NCSK (SEQ. ID. No. 835) 3 237-240 NQSG (SEQ. ID. No. 836)cAMP- and cGMP-dependent protein kinase phosphorylation site 170-173KRFT (SEQ. ID. No. 837) Protein kinase C phosphorylation site Number ofmatches: 7 1 89-91 SLK 2 164-166 SSK 3 383-385 SSK 4 169-171 TKR 5320-322 TFR 6 367-369 SGK 7 164-166 SSK Casein kinase II phosphorylationsite Number of matches: 5 1 27-30 SAFD (SEQ. ID. No. 838) 2 75-78 SLLD(SEQ. ID. No. 839) 3 258-261 SGCE (SEQ. ID. No. 840) 4 356-359 SVED(SEQ. ID. No. 841) 5 384-387 SKWE (SEQ. ID. No. 842) N-myristoylationsite Number of matches: 6 1 64-69 GAHPTL (SEQ. ID. No. 843) 2 131-136GGYIAN (SEQ. ID. No. 844) 3 225-230 GVAVNV (SEQ. ID. No. 845) 4 259-264GCERNH (SEQ. ID. No. 846) 5 287-292 GVLIAA (SEQ. ID. No. 847) 6 402-407GMYRCT (SEQ. ID. No. 848) Leucine zipper pattern 69-90LQADDDSLLDQDLPLTNSQLSL (SEQ. ID. No. 849)

[0848] TABLE XX Frequently Occurring Motifs avrg. % Name identityDescription Potential Function zf-C2H2 34% Zinc finger, C2H2 typeNucleic acid-binding protein functions as transcription factor, nuclearlocation probable cytochrome_b_N 68% Cytochrome b(N- membrane boundoxidase, generate terminal)/b6/petB superoxide ig 19% Immunoglobulindomain domains are one hundred amino acids long and include a conservedintradomain disulfide bond. WD40 18% WD domain, G-beta repeat tandemrepeats of about 40 residues, each containing a Trp-Asp motif. Functionin signal transduction and protein interaction PDZ 23% PDZ domain mayfunction in targeting signaling molecules to sub-membranous sites LRR28% Leucine Rich Repeat short sequence motifs involved in protein-protein interactions pkinase 23% Protein kinase domain conservedcatalytic core common to both serine/threonine and tyrosine proteinkinases containing an ATP binding site and a catalytic site PH 16% PHdomain pleckstrin homology involved in intracellular signaling or asconstituents of the cytoskeleton EGF 34% EGF-like domain 30-40amino-acid long found in the extracellular domain of membrane-boundproteins or in secreted proteins rvt 49% Reverse transcriptase(RNA-dependent DNA polymerase) ank 25% Ank repat Cytoplasmic protein,associates integral membrane proteins to the cytoskeleton oxidored_q132% NADH- membrane associated. Involved in protonUbiquinone/plastoquinone translocation across the membrane complex I),various chains efhand 24% EF hand calcium-binding domain, consists ofa12 residue loop flanked on both sides by a 12 residue alpha-helicaldomain rvp 79% Retroviral aspartyl protease Aspartyl or acid proteases,centered on a catalytic aspartyl residue Collagen 42% Collagen triplehelix repeat extracellular structural proteins involved in (20 copies)formation of connective tissue. The sequence consists of the G-X-Y andthe polypeptide chains forms a triple helix. fn3 20% Fibronectin typeIII domain Located in the extracellular ligand-binding region ofreceptors and is about 200 amino acid residues long with two pairs ofcysteines involved in disulfide bonds 7tm_1 19% 7 transmembrane receptorseven hydrophobic transmembrane regions, (rhodopsin family) with theN-terminus located extracellularly while the C-terminus is cytoplasmic.Signal through G proteins

[0849] TABLE XXI Properties of 108P5H8 Motifs and localization apply to108P5H8 variants 1 and 2. Bioinformatic Program URL Outcome ORF ORFFinder http://www.ncbi.nlm.gov/gorf 1290 (includes stop) Protein Lengthn/a n/a 429 amino acids Transmembrane TM Pred http://www.ch.embnet.org/6 TM, at amino acids region 114-130, 147-163, 181-200, 217-236, 273-295,306-324 HMMTop http://www.enzim.hu/hmmtop/ 6 TM, at amino acid 113-130,135-164, 179-202, 215-236, 271-296, 306-331 Sosuihttp://www.genome.ad.jp/SOSui/ 6 TM, at amino acid 113-135, 141-163,180-202, 215-237, 272-294, 308-330 TMHMMhttp://www.cbs.dtu.dk/services/TMHMM 6 TM, at amino acids 114-136,146-165, 178-200, 215-237, 273-295, 310-332 Signal Peptide Signal Phttp://www.cbs.dtu.dk/services/SignalP/ indicates no signal pI pI/MWtool http://www.expasy.ch/tools/ pI 6.11 Molecular weight pI/MW toolhttp://www.expasy.ch/tools/ 47.5 kDa Localization PSORThttp://psort.nibb.ac.jp/ Plasma membrane 60% PSORT IIhttp://psort.nibb.ac.jp/ Plasma membrane 43% iPSORThttp://psort.nibb.ac.jp No signal motif Motifs Pfamhttp://www.sanger.ac.uk/Pfam/ Ribosomal protein L34; Cation effluxfamily Prints http://www.biochem.ucl.ac.uk/ Rhodopsin Blockshttp://www.blocks.fhcrc.org/ No motif Prosite http://www.genome.ad.jp/No motif

[0850] TABLE XXII MHC Class 1 nonamer and decamer analysis of 108P5H8for selected alleles. Listed are scores that fall within the top 50%(rounded up) of all scores for the selected allele. SEQ. ID. Pos 1 2 3 45 6 7 8 9 score No. HLA-A*0201 nonamers 278 A L G D L V Q S V 30 1968121 Y L L F M I G E L 29 1969 153 A I I L T L L A L 28 1970 137 S L A IM T D A L 27 1971  75 S L L D Q D L P L 26 1972 150 D L S A I I L T L 261973 268 S L A V R A A F V 26 1974 323 I I W D T V V I I 26 1975 115 T IA A V L Y L L 25 1976 140 I M T D A L H M L 25 1977 181 V L S A M I S VL 25 1978 218 M L I T A A V G V 25 1979 343 Y I K E A L M K I 25 1980114 L T I A A V L Y L 23 1981 122 L L F M I G E L V 23 1982 146 H M L TD L S A I 23 1983 155 I L T L L A L W L 23 1984 185 M I S V L L V Y I 231985 198 L L Y E A V Q R T 23 1986 216 D I M L I T A A V 23 1987 281 D LV Q S V G V L 23 1988 330 I I L E G V P S H 23 1989 331 I L E G V P S HL 23 1990 409 Q L Q S Y R Q E V 23 1991  13 M L R K D D A P L 22 1992 68 T L Q A D D D S L 22 1993 111 K A R L T I A A V 22 1994 133 Y I A NS L A I M 22 1995 183 S A M I S V L L V 22 1996 306 P I C T Y V F S L 221997 322 R I I W D T V V I 22 1998 402 G M Y R C T I Q L 22 1999  76 L LD Q D L P L T 21 2000 147 M L T D L S A I I 21 2001 193 I L M G F L L YE 21 2002 194 L M G F L L Y E A 21 2003 220 I T A A V G V A V 21 2004349 M K I E D V Y S V 21 2005 118 A V L Y L L F M I 20 2006 124 F M I GE L V G G 20 2007 158 L L A L W L S S K 20 2008 178 R L E V L S A M I 202009 212 E I N G D I M L I 20 2010 222 A A V G V A V N V 20 2011 271 V RA A F V H A L 20 2012  80 D L P L T N S Q L 19 2013  82 P L T N S Q L SL 19 2014 104 I L K Q R K V K A 19 2015 143 D A L H M L T D L 19 2016154 I I L T L L A L W 19 2017 188 V L L V Y I L M G 19 2018 223 A V G VA V N V I 19 2019 302 K I A D P I C T Y 19 2020 327 T V V I I L E G V 192021 364 S L T S G K S T A 19 2022 395 H L L L N T F G M 19 2023  41 S RF N K L R V V 18 2024 151 L S A I I L T L L 18 2025 186 I S V L L V Y IL 18 2026 190 L V Y I L M G F L 18 2027 230 V I M G F L L N Q 18 2028303 I A D P I C T Y V 18 2029 346 E A L M K I E D V 18 2030 365 L T S GK S T A I 18 2031 370 S T A I V H I Q L 18 2032 371 T A I V H I Q L I 182033 377 Q L I P G S S S K 18 2034  45 K L R V V V A D D 17 2035  61 P VN G A H P T L 17 2036  87 Q L S L K V D S C 17 2037 184 A M I S V L L VY 17 2038 205 R T I H M N Y E I 17 2039 219 L I T A A V G V A 17 2040227 A V N V I M G F L 17 2041 289 L I A A Y I I R F 17 2042 308 C T Y VF S L L V 17 2043 315 L V A F T T F R I 17 2044 357 V E D L N I W S L 172045 112 A R L T I A A V L 16 2046 117 A A V L Y L L F M 16 2047 125 M IG E L V G G Y 16 2048 139 A I M T D A L H M 16 2049 148 L T D L S A I IL 16 2050 152 S A I I L T L L A 16 2051 157 T L L A L W L S S 16 2052174 F G F H R L E V L 16 2053 182 L S A M I S V L L 16 2054 189 L L V YI L M G F 16 2055 211 Y E I N G D I M L 16 2056 274 A F V H A L G D L 162057 275 F V H A L G D L V 16 2058 280 G D L V Q S V G V 16 2059 282 L VQ S V G V L I 16 2060 287 G V L I A A Y I I 16 2061 293 Y I I R F K P EY 16 2062 313 S L L V A F T T F 16 2063 324 I W D T V V I I L 16 2064392 K A N H L L L N T 16 2065  5 G A W K R L K S M 15 2066  9 R L K S ML R K D 15 2067  22 F L N D T S A F D 15 2068  27 S A F D F S D E A 152069  69 L Q A D D D S L L 15 2070 102 R E I L K Q R K V 15 2071 129 L VG G Y I A N S 15 2072 144 A L H M L T D L S 15 2073 180 E V L S A M I SV 15 2074 192 Y I L M G F L L Y 15 2075 231 I M G F L L N Q S 15 2076248 S L P S N S P T R 15 2077 295 I R F K P E Y K I 15 2078 310 Y V F SL L V A F 15 2079 314 L L V A F T T F R 15 2080 368 G K S T A I V H I 152081 HLA-A*0203 nonamers  49 V V A D D G S E A 12 850  63 N G A H P T LQ A 12 851 110 V K A R L T I A A 12 852  27 S A F D F S D E A 11 853 152S A I I L T L L A 11 854  57 A P E R P V N G A 10 855 270 A V R A A F VH A 10 856  11 K S M L R K D D A 9 857  20 P L F L N D T S A 9 858  43 FN K L R V V V A 9 859 104 I L K Q R K V K A 9 860 109 K V K A R L T I A9 861 127 G E L V G G Y I A 9 862 131 G G Y I A N S L A 9 863 136 N S LA I M T D A 9 864 145 L H M L T D L S A 9 865 176 F H R L E V L S A 9866 194 L M G F L L Y E A 9 867 214 N G D I M L I T A 9 868 215 G D I ML I T A A 9 869 219 L I T A A V G V A 9 870 262 R N H G Q D S L A 9 871265 G Q D S L A V R A 9 872 266 Q D S L A V R A A 9 873 283 V Q S V G VL I A 9 874 284 Q S V G V L I A A 9 875 296 R F K P E Y K I A 9 876 309T Y V F S L L V A 9 877 339 L N V D Y I K E A 9 878 364 S L T S G K S TA 9 879 385 K W E E V Q S K A 9 880 414 R Q E V D R T C A 9 881 HLA-A1nonamers 192 Y I L M G F L L Y 30 882 184 A M I S V L L V Y 25 883 148 LT D L S A I I L 20 884 396 L L L N T F G M Y 20 885  70 Q A D D D S L LD 18 886 113 R L T I A A V L Y 18 887 141 M T D A L H M L T 18 888 347 AL M K I E D V Y 18 889  71 A D D D S L L D Q 17 890 285 S V G V L I A AY 17 891 293 Y I I R F K P E Y 17 892 324 I W D T V V I I L 17 893 335 VP S H L N V D Y 17 894 356 S V E D L N I W S 17 895 405 R C T I Q L Q SY 17 896  15 R K D D A P L F L 16 897  31 F S D E A G D E G 16 898 125 MI G E L V G G Y 16 899 203 V Q R T I H M N Y 16 900 302 K I A D P I C TY 16 901  54 G S E A P E R P V 15 902 HLA-A26 nonamers 310 Y V F S L L VA F 30 903 125 M I G E L V G G Y 28 904 281 D L V Q S V G V L 28 905 285S V G V L I A A Y 27 906 388 E V Q S K A N H L 27 907 150 D L S A I I LT L 26 908 289 L I A A Y I I R F 26 909 302 K I A D P I C T Y 26 910  80D L P L T N S Q L 25 911 133 Y I A N S L A I M 25 912 153 A I I L T L LA L 25 913 114 L T I A A V L Y L 24 914 189 L L V Y I L M G F 24 915 192Y I L M G F L L Y 24 916 293 Y I I R F K P E Y 24 917 115 T I A A V L YL L 23 918 190 L V Y I L M G F L 23 919 212 E I N G D I M L I 23 920 306P I C T Y V F S L 23 921 396 L L L N T F G M Y 23 922 168 P T K R F T FG F 22 923 180 E V L S A M I S V 22 924 187 S V L L V Y I L M 22 925 227A V N V I M G F L 22 926 313 S L L V A F T T F 22 927  61 P V N G A H PT L 21 928 121 Y L L F M I G E L 21 929 128 E L V G G Y I A N 21 930 181V L S A M I S V L 21 931 326 D T V V I I L E G 21 932  38 E G L S R F NK L 20 933 148 L T D L S A I I L 20 934 331 I L E G V P S H L 20 935 340N V D Y I K E A L 20 936 370 S T A I V H I Q L 20 937  35 A G D E G L SR F 19 938 113 R L T I A A V L Y 19 939 139 A I M T D A L H M 19 940 171R F T F G F H R L 19 941 343 Y I K E A L M K I 19 942 347 A L M K I E DV Y 19 943 352 E D V Y S V E D L 19 944 416 E V D R T C A N C 19 945  13M L R K D D A P L 18 946  21 L F L N D T S A F 18 947  75 S L L D Q D LP L 18 948  82 P L T N S Q L S L 18 949 103 E I L K Q R K V K 18 950 129L V G G Y I A N S 18 951 137 S L A I M T D A L 18 952 143 D A L H M L TD L 18 953 216 D I M L I T A A V 18 954 267 D S L A V R A A F 18 955 274A F V H A L G D L 18 956 330 I I L E G V P S H 18 957 399 N T F G M Y RC T 18 958  25 D T S A F D F S D 17 959  68 T L Q A D D D S L 17 960  83L T N S Q L S L K 17 961 155 I L T L L A L W L 17 962 184 A M I S V L LV Y 17 963 230 V I M G F L L N Q 17 964 353 D V Y S V E D L N 17 965 395H L L L N T F G M 17 966 405 R C T I Q L Q S Y 17 967  73 D D S L L D QD L 16 968 116 I A A V L Y L L F 16 969 118 A V L Y L L F M I 16 970 154I I L T L L A L W 16 971 177 H R L E V L S A M 16 972 185 M I S V L L VY I 16 973 198 L L Y E A V Q R T 16 974 201 E A V Q R T I H M 16 975 225G V A V N V I M G 16 976 226 V A V N V I M G F 16 977 229 N V I M G F LL N 16 978 270 A V R A A F V H A 16 979 278 A L G D L V Q S V 16 980 319T T F R I I W D T 16 981 323 I I W D T V V I I 16 982 334 G V P S H L NV D 16 983 350 K I E D V Y S V E 16 984 378 L I P G S S S K W 16 985  9R L K S M L R K D 15 986  17 D D A P L F L N D 15 987  76 L L D Q D L PL T 15 988 109 K V K A R L T I A 15 989 158 L L A L W L S S K 15 990 202A V Q R T I H M N 15 991 261 E R N H G Q D S L 15 992 327 T V V I I L EG V 15 993 373 I V H I Q L I P G 15 994 393 A N H L L L N T F 15 995 406C T I Q L Q S Y R 15 996 HLA-A3 nonamers 377 Q L I P G S S S K 35 997113 R L T I A A V L Y 28 998 158 L L A L W L S S K 27 999 197 F L L Y EA V Q R 26 1000 103 E I L K Q R K V K 25 1001 162 W L S S K S P T K 251002 294 I I R F K P E Y K 25 1003 302 K I A D P I C T Y 23 1004 322 R II W D T V V I 23 1005 347 A L M K I E D V Y 23 1006 270 A V R A A F V HA 22 1007 313 S L L V A F T T F 22 1008 192 Y I L M G F L L Y 21 1009285 S V G V L I A A Y 21 1010 330 I I L E G V P S H 21 1011 361 N I W SL T S G K 21 1012 396 L L L N T F G M Y 21 1013  45 K L R V V V A D D 201014 155 I L T L L A L W L 20 1015 184 A M I S V L L V Y 20 1016 218 M LI T A A V G V 20 1017 268 S L A V R A A F V 20 1018 293 Y I I R F K P EY 20 1019 342 D Y I K E A L M K 20 1020 350 K I E D V Y S V E 20 1021364 S L T S G K S T A 20 1022 104 I L K Q R K V K A 19 1023 109 K V K AR L T I A 19 1024 150 D L S A I I L T L 19 1025 153 A I I L T L L A L 191026 157 T L L A L W L S S 19 1027 181 V L S A M I S V L 19 1028 248 S LP S N S P T R 19 1029 288 V L I A A Y I I R 19 1030 310 Y V F S L L V AF 19 1031 328 V V I I L E G V P 19 1032 329 V I I L E G V P S 19 1033331 I L E G V P S H L 19 1034  8 K R L K S M L R K 18 1035  49 V V A D DG S E A 18 1036 178 R L E V L S A M I 18 1037 229 N V I M G F L L N 181038 234 F L L N Q S G H R 18 1039 282 L V Q S V G V L I 18 1040 359 D LN I W S L T S 18 1041 397 L L N T F G M Y R 18 1042  39 G L S R F N K LR 17 1043  75 S L L D Q D L P L 17 1044  80 D L P L T N S Q L 17 1045139 A I M T D A L H M 17 1046 193 I L M G F L L Y E 17 1047 202 A V Q RT I H M N 17 1048 223 A V G V A V N V I 17 1049 235 L L N Q S G H R H 171050 314 L L V A F T T F R 17 1051 411 Q S Y R Q E V D R 17 1052HLA-B*0702 nonamers 57 A P E R P V N G A 22 1053  60 R P V N G A H P T18 1054  2 A G S G A W K R L 16 1055  13 M L R K D D A P L 15 1056  15 RK D D A P L F L 15 1057 106 K Q R K V K A R L 15 1058 150 D L S A I I LT L 15 1059 153 A I I L T L L A L 15 1060  97 N C S K Q R E I L 14 1061112 A R L T I A A V L 14 1062 114 L T I A A V L Y L 14 1063 167 S P T KR F T F G 14 1064 181 V L S A M I S V L 14 1065 270 A V R A A F V H A 141066 271 V R A A F V H A L 14 1067 335 V P S H L N V D Y 14 1068  61 P VN G A H P T L 13 1069  63 N G A H P T L Q A 13 1070  75 S L L D Q D L PL 13 1071 137 S L A I M T D A L 13 1072 155 I L T L L A L W L 13 1073182 L S A M I S V L L 13 1074 220 I T A A V G V A V 13 1075 227 A V N VI M G F L 13 1076 249 L P S N S P T R G 13 1077 305 D P I C T Y V F S 131078 324 I W D T V V I I L 13 1079 331 I L E G V P S H L 13 1080 389 V QS K A N H L L 13 1081 390 Q S K A N H L L L 13 1082  6 A W K R L K S M L12 1083  19 A P L F L N D T S 12 1084  38 E G L S R F N K L 12 1085  42R F N K L R V V V 12 1086  73 D D S L L D Q D L 12 1087  82 P L T N S QL S L 12 1088 115 T I A A V L Y L L 12 1089 151 L S A I I L T L L 121090 171 R F T F G F H R L 12 1091 186 I S V L L V Y I L 12 1092 222 A AV G V A V N V 12 1093 274 A F V H A L G D L 12 1094 281 D L V Q S V G VL 12 1095 298 K P E Y K I A D P 12 1096 307 I C T Y V F S L L 12 1097340 N V D Y I K E A L 12 1098 352 E D V Y S V E D L 12 1099 365 L T S GK S T A I 12 1100 379 I P G S S S K W E 12 1101  32 S D E A G D E G L 111102  54 G S E A P E R P V 11 1103  68 T L Q A D D D S L 11 1104  69 L QA D D D S L L 11 1105 108 R K V K A R L T I 11 1106 111 K A R L T I A AV 11 1107 117 A A V L Y L L F M 11 1108 130 V G G Y I A N S L 11 1109139 A I M T D A L H M 11 1110 140 I M T D A L H M L 11 1111 143 D A L HM L T D L 11 1112 148 L T D L S A I I L 11 1113 174 F G F H R L E V L 111114 176 F H R L E V L S A 11 1115 190 L V Y I L M G F L 11 1116 191 V YI L M G F L L 11 1117 223 A V G V A V N V I 11 1118 228 V N V I M G F LL 11 1119 261 E R N H G Q D S L 11 1120 283 V Q S V G V L I A 11 1121295 I R F K P E Y K I 11 1122 306 P I C T Y V F S L 11 1123 311 V F S LL V A F T 11 1124 322 R I I W D T V V I 11 1125 357 V E D L N I W S L 111126 370 S T A I V H I Q L 11 1127 388 E V Q S K A N H L 11 1128 402 G MY R C T I Q L 11 1129 HLA-B*08 nonamers 104 I L K Q R K V K A 26 1130 13 M L R K D D A P L 22 1131  43 F N K L R V V V A 22 1132 343 Y I K EA L M K I 22 1133 388 E V Q S K A N H L 22 1134 107 Q R K V K A R L T 211135 294 I I R F K P E Y K 21 1136  6 A W K R L K S M L 20 1137  38 E GL S R F N K L 20 1138 390 Q S K A N H L L L 20 1139  89 S L K V D S C DN 19 1140 137 S L A I M T D A L 19 1141 164 S S K S P T K R F 19 1142174 F G F H R L E V L 19 1143  75 S L L D Q D L P L 18 1144  80 D L P LT N S Q L 18 1145  87 Q L S L K V D S C 18 1146  98 C S K Q R E I L K 181147 109 K V K A R L T I A 18 1148 121 Y L L F M I G E L 18 1149 155 I LT L L A L W L 18 1150 181 V L S A M I S V L 18 1151 298 K P E Y K I A DP 18 1152  7 W K R L K S M L R 17 1153  56 E A P E R P V N G 17 1154  96D N C S K Q R E I 17 1155 150 D L S A I I L T L 17 1156 268 S L A V R AA F V 17 1157 281 D L V Q S V G V L 17 1158 331 I L E G V P S H L 171159 346 E A L M K I E D V 17 1160 365 L T S G K S T A I 17 1161  9 R LK S M L R K D 16 1162  14 L R K D D A P L F 16 1163  68 T L Q A D D D SL 16 1164  82 P L T N S Q L S L 16 1165 106 K Q R K V K A R L 16 1166153 A I I L T L L A L 16 1167 162 W L S S K S P T K 16 1168 166 K S P TK R F T F 16 1169 167 S P T K R F T F G 16 1170 168 P T K R F T F G F 161171 306 P I C T Y V F S L 15 1172 313 S L L V A F T T F 15 1173  5 G AW K R L K S M 14 1174  45 K L R V V V A D D 14 1175 115 T I A A V L Y LL 14 1176 143 D A L H M L T D L 14 1177 201 E A V Q R T I H M 14 1178352 E D V Y S V E D L 14 1179  12 S M L R K D D A P 13 1180 105 L K Q RK V K A R 13 1181 111 K A R L T I A A V 13 1182 178 R L E V L S A M I 131183 186 I S V L L V Y I L 13 1184 189 L L V Y I L M G F 13 1185 212 E IN G D I M L I 13 1186 258 S G C E R N H G Q 13 1187 271 V R A A F V H AL 13 1188 323 I I W D T V V I I 13 1189 357 V E D L N I W S L 13 1190383 S S K W E E V Q S 13 1191 HLA-B*1510 nonamers 331 I L E G V P S H L15 1192  2 A G S G A W K R L 14 1193 106 K Q R K V K A R L 14 1194 181 VL S A M I S V L 14 1195 276 V H A L G D L V Q 14 1196 281 D L V Q S V GV L 14 1197  61 P V N G A H P T L 13 1198  97 N C S K Q R E I L 13 1199121 Y L L F M I G E L 13 1200 140 I M T D A L H M L 13 1201 150 D L S AI I L T L 13 1202 171 R F T F G F H R L 13 1203 182 L S A M I S V L L 131204 186 I S V L L V Y I L 13 1205 271 V R A A F V H A L 13 1206 324 I WD T V V I I L 13 1207 374 V H I Q L I P G S 13 1208  15 R K D D A P L FL 12 1209  32 S D E A G D E G L 12 1210  69 L Q A D D D S L L 12 1211112 A R L T I A A V L 12 1212 115 T I A A V L Y L L 12 1213 137 S L A IM T D A L 12 1214 155 I L T L L A L W L 12 1215 174 F G F H R L E V L 121216 207 I H M N Y E I N G 12 1217 211 Y E I N G D I M L 12 1218 227 A VN V I M G F L 12 1219 244 S H S H S L P S N 12 1220 246 S H S L P S N SP 12 1221 261 E R N H G Q D S L 12 1222 263 N H G Q D S L A V 12 1223307 I C T Y V F S L L 12 1224 340 N V D Y I K E A L 12 1225 352 E D V YS V E D L 12 1226 357 V E D L N I W S L 12 1227 389 V Q S K A N H L L 121228  6 A W K R L K S M L 11 1229  13 M L R K D D A P L 11 1230  38 E GL S R F N K L 11 1231  68 T L Q A D D D S L 11 1232  73 D D S L L D Q DL 11 1233  75 S L L D Q D L P L 11 1234 114 L T I A A V L Y L 11 1235151 L S A I I L T L L 11 1236 153 A I I L T L L A L 11 1237 176 F H R LE V L S A 11 1238 190 L V Y I L M G F L 11 1239 240 G H R H S H S H S 111240 242 R H S H S H S L P 11 1241 306 P I C T Y V F S L 11 1242 370 S TA I V H I Q L 11 1243 388 E V Q S K A N H L 11 1244 390 Q S K A N H L LL 11 1245 402 G M Y R C T I Q L 11 1246  35 A G D E G L S R F 10 1247 65 A H P T L Q A D D 10 1248  80 D L P L T N S Q L 10 1249  82 P L T NS Q L S L 10 1250 116 I A A V L Y L L F 10 1251 130 V G G Y I A N S L 101252 143 D A L H M L T D L 10 1253 145 L H M L T D L S A 10 1254 148 L TD L S A I I L 10 1255 191 V Y I L M G F L L 10 1256 228 V N V I M G F LL 10 1257 241 H R H S H S H S L 10 1258 274 A F V H A L G D L 10 1259289 L I A A Y I I R F 10 1260 310 Y V F S L L V A F 10 1261 337 S H L NV D Y I K 10 1262 394 N H L L L N T F G 10 1263 164 S S K S P T K R F 91264  5 G A W K R L K S M 8 1265  14 L R K D D A P L F 8 1266 133 Y I AN S L A I M 8 1267 166 K S P T K R F T F 8 1268 177 H R L E V L S A M 81269 201 E A V Q R T I H M 8 1270 210 N Y E I N G D I M 8 1271 224 V G VA V N V I M 8 1272 267 D S L A V R A A F 8 1273 304 A D P I C T Y V F 81274 313 S L L V A F T T F 8 1275  21 L F L N D T S A F 7 1276  55 S E AP E R P V N 7 1277 221 T A A V G V A V N 7 1278 226 V A V N V I M G F 71279 393 A N H L L L N T F 7 1280 395 H L L L N T F G M 7 1281HLA-B*2705 nonamers  8 K R L K S M L R K 30 1282 112 A R L T I A A V L27 1283 295 I R F K P E Y K I 26 1284 170 K R F T F G F H R 25 1285 261E R N H G Q D S L 25 1286 101 Q R E I L K Q R K 24 1287 177 H R L E V LS A M 24 1288  14 L R K D D A P L F 22 1289 241 H R H S H S H S L 221290 271 V R A A F V H A L 21 1291 106 K Q R K V K A R L 19 1292  35 A GD E G L S R F 18 1293 171 R F T F G F H R L 18 1294 377 Q L I P G S S SK 18 1295 406 C T I Q L Q S Y R 18 1296 163 L S S K S P T K R 17 1297233 G F L L N Q S G H 17 1298 330 I I L E G V P S H 17 1299 342 D Y I KE A L M K 17 1300 402 G M Y R C T I Q L 17 1301  1 M A G S G A W K R 161302  41 S R F N K L R V V 16 1303 100 K Q R E I L K Q R 16 1304 121 Y LL F M I G E L 16 1305 150 D L S A I I L T L 16 1306 153 A I I L T L L AL 16 1307 186 I S V L L V Y I L 16 1308 197 F L L Y E A V Q R 16 1309211 Y E I N G D I M L 16 1310 226 V A V N V I M G F 16 1311 256 R G S GC E R N H 16 1312 281 D L V Q S V G V L 16 1313 289 L I A A Y I I R F 161314 310 Y V F S L L V A F 16 1315 321 F R I I W D T V V 16 1316 357 V ED L N I W S L 16 1317 387 E E V Q S K A N H 16 1318 393 A N H L L L N TF 16 1319  2 A G S G A W K R L 15 1320  5 G A W K R L K S M 15 1321  15R K D D A P L F L 15 1322  38 E G L S R F N K L 15 1323  82 P L T N S QL S L 15 1324 103 E I L K Q R K V K 15 1325 108 R K V K A R L T I 151326 143 D A L H M L T D L 15 1327 155 I L T L L A L W L 15 1328 174 F GF H R L E V L 15 1329 181 V L S A M I S V L 15 1330 184 A M I S V L L VY 15 1331 205 R T I H M N Y E I 15 1332 290 I A A Y I I R F K 15 1333302 K I A D P I C T Y 15 1334 313 S L L V A F T T F 15 1335 331 I L E GV P S H L 15 1336 337 S H L N V D Y I K 15 1337 404 Y R C T I Q L Q S 151338 HLA-B*2709 nonamers 112 A R L T I A A V L 25 1339 295 I R F K P E YK I 22 1340  14 L R K D D A P L F 21 1341 241 H R H S H S H S L 21 1342271 V R A A F V H A L 21 1343  41 S R F N K L R V V 20 1344 177 H R L EV L S A M 20 1345 261 E R N H G Q D S L 20 1346 321 F R I I W D T V V 191347  8 K R L K S M L R K 16 1348 171 R F T F G F H R L 16 1349 402 G MY R C T I Q L 16 1350  15 R K D D A P L F L 15 1351 287 G V L I A A Y II 15 1352 322 R I I W D T V V I 15 1353 108 R K V K A R L T I 14 1354155 I L T L L A L W L 14 1355 170 K R F T F G F H R 14 1356 186 I S V LL V Y I L 14 1357 205 R T I H M N Y E I 14 1358 280 G D L V Q S V G V 141359  38 E G L S R F N K L 13 1360  42 R F N K L R V V V 13 1361  75 S LL D Q D L P L 13 1362 102 R E I L K Q R K V 13 1363 106 K Q R K V K A RL 13 1364 114 L T I A A V L Y L 13 1365 132 G Y I A N S L A I 13 1366153 A I I L T L L A L 13 1367 222 A A V G V A V N V 13 1368 404 Y R C TI Q L Q S 13 1369  2 A G S G A W K R L 12 1370  46 L R V V V A D D G 121371  82 P L T N S Q L S L 12 1372 121 Y L L F M I G E L 12 1373 130 V GG Y I A N S L 12 1374 139 A I M T D A L H M 12 1375 140 I M T D A L H ML 12 1376 143 D A L H M L T D L 12 1377 150 D L S A I I L T L 12 1378174 F G F H R L E V L 12 1379 178 R L E V L S A M I 12 1380 182 L S A MI S V L L 12 1381 190 L V Y I L M G F L 12 1382 227 A V N V I M G F L 121383 255 T R G S G C E R N 12 1384 274 A F V H A L G D L 12 1385 281 D LV Q S V G V L 12 1386 299 P E Y K I A D P I 12 1387 307 I C T Y V F S LL 12 1388 310 Y V F S L L V A F 12 1389 324 I W D T V V I I L 12 1390333 E G V P S H L N V 12 1391 349 M K I E D V Y S V 12 1392 352 E D V YS V E D L 12 1393 368 G K S T A I V H I 12 1394 381 G S S S K W E E V 121395 388 E V Q S K A N H L 12 1396 418 D R T C A N C Q S 12 1397HLA-B*5101 nonamers 316 V A F T T F R I I 26 1398 143 D A L H M L T D L25 1399 222 A A V G V A V N V 23 1400 183 S A M I S V L L V 22 1401 346E A L M K I E D V 22 1402 371 T A I V H I Q L I 22 1403 303 I A D P I CT Y V 21 1404 111 K A R L T I A A V 20 1405 126 I G E L V G G Y I 201406  38 E G L S R F N K L 18 1407 174 F G F H R L E V L 18 1408 199 L YE A V Q R T I 18 1409 209 M N Y E I N G D I 18 1410 286 V G V L I A A YI 18 1411 323 I I W D T V V I I 18 1412  18 D A P L F L N D T 17 1413343 Y I K E A L M K I 17 1414  96 D N C S K Q R E I 16 1415 159 L A L WL S S K S 16 1416 195 M G F L L Y E A V 16 1417 221 T A A V G V A V N 161418 223 A V G V A V N V I 16 1419 249 L P S N S P T R G 16 1420 269 L AV R A A F V H 16 1421 281 D L V Q S V G V L 16 1422 291 A A Y I I R F KP 16 1423 295 I R F K P E Y K I 16 1424 299 P E Y K I A D P I 16 1425305 D P I C T Y V F S 16 1426 333 E G V P S H L N V 16 1427  2 A G S G AW K R L 15 1428  19 A P L F L N D T S 15 1429  41 S R F N K L R V V 151430  56 E A P E R P V N G 15 1431  81 L P L T N S Q L S 15 1432 130 V GG Y I A N S L 15 1433 150 D L S A I I L T L 15 1434 277 H A L G D L V QS 15 1435 282 L V Q S V G V L I 15 1436 365 L T S G K S T A I 15 1437  1M A G S G A W K R 14 1438  5 G A W K R L K S M 14 1439  53 D G S E A P ER P 14 1440 108 R K V K A R L T I 14 1441 134 I A N S L A I M T 14 1442146 H M L T D L S A I 14 1443 220 I T A A V G V A V 14 1444 226 V A V NV I M G F 14 1445 290 I A A Y I I R F K 14 1446 315 L V A F T T F R I 141447 322 R I I W D T V V I 14 1448 324 I W D T V V I I L 14 1449 368 G KS T A I V H I 14 1450 379 I P G S S S K W E 14 1451 400 T F G M Y R C TI 14 1452  42 R F N K L R V V V 13 1453  57 A P E R P V N G A 13 1454 80 D L P L T N S Q L 13 1455 112 A R L T I A A V L 13 1456 116 I A A VL Y L L F 13 1457 117 A A V L Y L L F M 13 1458 118 A V L Y L L F M I 131459 138 L A I M T D A L H 13 1460 167 S P T K R F T F G 13 1461 181 V LS A M I S V L 13 1462 185 M I S V L L V Y I 13 1463 273 A A F V H A L GD 13 1464 279 L G D L V Q S V G 13 1465 287 G V L I A A Y I I 13 1466308 C T Y V F S L L V 13 1467 321 F R I I W D T V V 13 1468 331 I L E GV P S H L 13 1469 335 V P S H L N V D Y 13 1470 349 M K I E D V Y S V 131471 SEQ. ID. Pos 1 2 3 4 5 6 7 8 9 0 score No. HLA-A*0201 decamers 139A I M T D A L H M L 26 1472 184 A M I S V L L V Y I 26 1473 193 I L M GF L L Y E A 26 1474 323 I I W D T V V I I L 26 1475 348 L M K I E D V YS V 26 1476 198 L L Y E A V Q R T I 25 1477 217 I M L I T A A V G V 251478  12 S M L R K D D A P L 24 1479  75 S L L D Q D L P L T 24 1480 113R L T I A A V L Y L 24 1481 152 S A I I L T L L A L 24 1482 277 H A L GD L V Q S V 24 1483 322 R I I W D T V V I I 24 1484 330 I I L E G V P SH L 24 1485 114 L T I A A V L Y L L 23 1486 121 Y L L F M I G E L V 231487 150 D L S A I I L T L L 23 1488 154 I I L T L L A L W L 23 1489 181V L S A M I S V L L 23 1490 185 M I S V L L V Y I L 23 1491  39 G L S RF N K L R V 22 1492 219 L I T A A V G V A V 22 1493 270 A V R A A F V HA L 22 1494 302 K I A D P I C T Y V 22 1495 356 S V E D L N I W S L 221496 129 L V G G Y I A N S L 21 1497 172 F T F G F H R L E V 21 1498 189L L V Y I L M G F L 21 1499 222 A A V G V A V N V I 21 1500 273 A A F VH A L G D L 21 1501  68 T L Q A D D D S L L 20 1502  83 L T N S Q L S LK V 20 1503 110 V K A R L T I A A V 20 1504 120 L Y L L F M I G E L 201505 146 H M L T D L S A I I 20 1506 147 M L T D L S A I I L 20 1507 194L M G F L L Y E A V 20 1508 197 F L L Y E A V Q R T 20 1509 218 M L I TA A V G V A 20 1510 294 I I R F K P E Y K I 20 1511 314 L L V A F T T FR I 20 1512 338 H L N V D Y I K E A 20 1513 364 S L T S G K S T A I 201514 365 L T S G K S T A I V 20 1515 370 S T A I V H I Q L I 20 1516 144A L H M L T D L S A 19 1517 149 T D L S A I I L T L 19 1518 182 L S A MI S V L L V 19 1519 281 D L V Q S V G V L I 19 1520 117 A A V L Y L L FM I 18 1521 124 F M I G E L V G G Y 18 1522 125 M I G E L V G G Y I 181523 153 A I I L T L L A L W 18 1524 157 T L L A L W L S S K 18 1525 188V L L V Y I L M G F 18 1526 221 T A A V G V A V N V 18 1527 326 D T V VI I L E G V 18 1528 329 V I I L E G V P S H 18 1529  81 L P L T N S Q LS L 17 1530 111 K A R L T I A A V L 17 1531 116 I A A V L Y L L F M 171532 160 A L W L S S K S P T 17 1533 180 E V L S A M I S V L 17 1534 190L V Y I L M G F L L 17 1535 192 Y I L M G F L L Y E 17 1536 227 A V N VI M G F L L 17 1537 230 V I M G F L L N Q S 17 1538 280 G D L V Q S V GV L 17 1539 288 V L I A A Y I I R F 17 1540 305 D P I C T Y V F S L 171541 310 Y V F S L L V A F T 17 1542 319 T T F R I I W D T V 17 1543 345K E A L M K I E D V 17 1544 399 N T F G M Y R C T I 17 1545  5 G A W K RL K S M L 16 1546 133 Y I A N S L A I M T 16 1547 137 S L A I M T D A LH 16 1548 142 T D A L H M L T D L 16 1549 145 L H M L T D L S A I 161550 158 L L A L W L S S K S 16 1551 208 H M N Y E I N G D I 16 1552 211Y E I N G D I M L I 16 1553 215 G D I M L I T A A V 16 1554 285 S V G VL I A A Y I 16 1555 289 L I A A Y I I R F K 16 1556 351 I E D V Y S V ED L 16 1557 359 D L N I W S L T S G 16 1558 377 Q L I P G S S S K W 161559 408 I Q L Q S Y R Q E V 16 1560  40 L S R F N K L R V V 15 1561  41S R F N K L R V V V 15 1562  67 P T L Q A D D D S L 15 1563  76 L L D QD L P L T N 15 1564 103 E I L K Q R K V K A 15 1565 104 I L K Q R K V KA R 15 1566 128 E L V G G Y I A N S 15 1567 155 I L T L L A L W L S 151568 226 V A V N V I M G F L 15 1569 279 L G D L V Q S V G V 15 1570 282L V Q S V G V L I A 15 1571 306 P I C T Y V F S L L 15 1572 315 L V A FT T F R I I 15 1573 342 D Y I K E A L M K I 15 1574 347 A L M K I E D VY S 15 1575 367 S G K S T A I V H I 15 1576  31 F S D E A G D E G L 141577  79 Q D L P L T N S Q L 14 1578 105 L K Q R K V K A R L 14 1579 122L L F M I G E L V G 14 1580 140 I M T D A L H M L T 14 1581 148 L T D LS A I I L T 14 1582 177 H R L E V L S A M I 14 1583 179 L E V L S A M IS V 14 1584 220 I T A A V G V A V N 14 1585 234 F L L N Q S G H R H 141586 240 G H R H S H S H S L 14 1587 262 R N H G Q D S L A V 14 1588 268S L A V R A A F V H 14 1589 308 C T Y V F S L L V A 14 1590 313 S L L VA F T T F R 14 1591 331 I L E G V P S H L N 14 1592 339 L N V D Y I K EA L 14 1593 372 A I V H I Q L I P G 14 1594 396 L L L N T F G M Y R 141595 397 L L N T F G M Y R C 14 1596 402 G M Y R C T I Q L Q 14 1597  1M A G S G A W K R L 13 1598  4 S G A W K R L K S M 13 1599  14 L R K D DA P L F L 13 1600  22 F L N D T S A F D F 13 1601  56 E A P E R P V N GA 13 1602  60 R P V N G A H P T L 13 1603 119 V L Y L L F M I G E 131604 132 G Y I A N S L A I M 13 1605 134 I A N S L A I M T D 13 1606 136N S L A I M T D A L 13 1607 138 L A I M T D A L H M 13 1608 170 K R F TF G F H R L 13 1609 173 T F G F H R L E V L 13 1610 187 S V L L V Y I LM G 13 1611 212 E I N G D I M L I T 13 1612 213 I N G D I M L I T A 131613 235 L L N Q S G H R H S 13 1614 267 D S L A V R A A F V 13 1615 278A L G D L V Q S V G 13 1616 321 F R I I W D T V V I 13 1617 332 L E G VP S H L N V 13 1618 335 V P S H L N V D Y I 13 1619 350 K I E D V Y S VE D 13 1620 353 D V Y S V E D L N I 13 1621 391 S K A N H L L L N T 131622 392 K A N H L L L N T F 13 1623 HLA-A*0203 decamers 109 K V K A R LT I A A 19 1624 214 N G D I M L I T A A 19 1625 265 G Q D S L A V R A A19 1626 283 V Q S V G V L I A A 19 1627 110 V K A R L T I A A V 17 1628215 G D I M L I T A A V 17 1629 266 Q D S L A V R A A F 17 1630 284 Q SV G V L I A A Y 17 1631  10 L K S M L R K D D A 10 1632  19 A P L F L ND T S A 10 1633  26 T S A F D F S D E A 10 1634  42 R F N K L R V V V A10 1635  48 V V V A D D G S E A 10 1636  56 E A P E R P V N G A 10 1637 62 V N G A H P T L Q A 10 1638 103 E I L K Q R K V K A 10 1639 108 R KV K A R L T I A 10 1640 126 I G E L V G G Y I A 10 1641 130 V G G Y I AN S L A 10 1642 135 A N S L A I M T D A 10 1643 144 A L H M L T D L S A10 1644 151 L S A I I L T L L A 10 1645 175 G F H R L E V L S A 10 1646193 I L M G F L L Y E A 10 1647 213 I N G D I M L I T A 10 1648 218 M LI T A A V G V A 10 1649 261 E R N H G Q D S L A 10 1650 264 H G Q D S LA V R A 10 1651 269 L A V R A A F V H A 10 1652 282 L V Q S V G V L I A10 1653 295 I R F K P E Y K I A 10 1654 308 C T Y V F S L L V A 10 1655338 H L N V D Y I K E A 10 1656 363 W S L T S G K S T A 10 1657 384 S KW E E V Q S K A 10 1658 413 Y R Q E V D R T C A 10 1659  11 K S M L R KD D A P 9 1660  20 P L F L N D T S A F 9 1661  27 S A F D F S D E A G 91662  43 F N K L R V V V A D 9 1663  49 V V A D D G S E A P 9 1664  57 AP E R P V N G A H 9 1665  63 N G A H P T L Q A D 9 1666 104 I L K Q R KV K A R 9 1667 127 G E L V G G Y I A N 9 1668 131 G G Y I A N S L A I 91669 136 N S L A I M T D A L 9 1670 145 L H M L T D L S A I 9 1671 152 SA I I L T L L A L 9 1672 176 F H R L E V L S A M 9 1673 194 L M G F L LY E A V 9 1674 219 L I T A A V G V A V 9 1675 262 R N H G Q D S L A V 91676 270 A V R A A F V H A L 9 1677 296 R F K P E Y K I A D 9 1678 309 TY V F S L L V A F 9 1679 339 L N V D Y I K E A L 9 1680 364 S L T S G KS T A I 9 1681 385 K W E E V Q S K A N 9 1682 414 R Q E V D R T C A N 91683 HLA-A1 decamers 191 V Y I L M G F L L Y 27 1684 183 S A M I S V L LV Y 24 1685 141 M T D A L H M L T D 22 1686 148 L T D L S A I I L T 221687 284 Q S V G V L I A A Y 20 1688 395 H L L L N T F G M Y 20 1689  16K D D A P L F L N D 19 1690 112 A R L T I A A V L Y 18 1691 124 F M I GE L V G G Y 18 1692  76 L L D Q D L P L T N 17 1693 301 Y K I A D P I CT Y 17 1694 404 Y R C T I Q L Q S Y 17 1695  54 G S E A P E R P V N 161696  70 Q A D D D S L L D Q 16 1697 202 A V Q R T I H M N Y 16 1698 292A Y I I R F K P E Y 16 1699 324 I W D T V V I I L E 16 1700 334 G V P SH L N V D Y 16 1701 346 E A L M K I E D V Y 16 1702  31 F S D E A G D EG L 15 1703 172 F T F G F H R L E V 15 1704 210 N Y E I N G D I M L 151705 303 I A D P I C T Y V F 15 1706  32 S D E A G D E G L S 14 1707  83L T N S Q L S L K V 14 1708 331 I L E G V P S H L N 14 1709 344 I K E AL M K I E D 14 1710  3 G S G A W K R L K S 13 1711  23 L N D T S A F D FS 13 1712 182 L S A M I S V L L V 13 1713 308 C T Y V F S L L V A 131714 357 V E D L N I W S L T 13 1715 HLA-A26 decamers 180 E V L S A M IS V L 30 1716 225 G V A V N V I M G F 27 1717 150 D L S A I I L T L L 261718 115 T I A A V L Y L L F 25 1719 288 V L I A A Y I I R F 25 1720 388E V Q S K A N H L L 25 1721 114 L T I A A V L Y L L 24 1722 188 V L L VY I L M G F 24 1723 356 S V E D L N I W S L 24 1724  34 E A G D E G L SR F 23 1725 139 A I M T D A L H M L 23 1726 270 A V R A A F V H A L 231727 306 P I C T Y V F S L L 23 1728 323 I I W D T V V I I L 23 1729 334G V P S H L N V D Y 23 1730 395 H L L L N T F G M Y 23 1731  20 P L F LN D T S A F 22 1732 128 E L V G G Y I A N S 22 1733 185 M I S V L L V YI L 22 1734 202 A V Q R T I H M N Y 22 1735 212 E I N G D I M L I T 221736 330 I I L E G V P S H L 22 1737  25 D T S A F D F S D E 21 1738 129L V G G Y I A N S L 21 1739 305 D P I C T Y V F S L 21 1740 326 D T V VI I L E G V 21 1741  13 M L R K D D A P L F 20 1742  67 P T L Q A D D DS L 20 1743 113 R L T I A A V L Y L 20 1744 189 L L V Y I L M G F L 201745 340 N V D Y I K E A L M 20 1746  22 F L N D T S A F D F 19 1747  37D E G L S R F N K L 19 1748 103 E I L K Q R K V K A 19 1749 124 F M I GE L V G G Y 19 1750 223 A V G V A V N V I M 19 1751 359 D L N I W S L TS G 19 1752 154 I I L T L L A L W L 18 1753 173 T F G F H R L E V L 181754 190 L V Y I L M G F L L 18 1755 227 A V N V I M G F L L 18 1756 284Q S V G V L I A A Y 18 1757 310 Y V F S L L V A F T 18 1758 329 V I I LE G V P S H 18 1759 353 D V Y S V E D L N I 18 1760  68 T L Q A D D D SL L 17 1761  72 D D D S L L D Q D L 17 1762 147 M L T D L S A I I L 171763 153 A I I L T L L A L W 17 1764 181 V L S A M I S V L L 17 1765 216D I M L I T A A V G 17 1766 229 N V I M G F L L N Q 17 1767 230 V I M GF L L N Q S 17 1768 322 R I I W D T V V I I 17 1769 343 Y I K E A L M KI E 17 1770 416 E V D R T C A N C Q 17 1771  80 D L P L T N S Q L S 161772 191 V Y I L M G F L L Y 16 1773 205 R T I H M N Y E I N 16 1774 220I T A A V G V A V N 16 1775 281 D L V Q S V G V L I 16 1776 301 Y K I AD P I C T Y 16 1777 309 T Y V F S L L V A F 16 1778 346 E A L M K I E DV Y 16 1779 370 S T A I V H I Q L I 16 1780 373 I V H I Q L I P G S 161781 387 E E V Q S K A N H L 16 1782 404 Y R C T I Q L Q S Y 16 1783  96D N C S K Q R E I L 15 1784 104 I L K Q R K V K A R 15 1785 118 A V L YL L F M I G 15 1786 132 G Y I A N S L A I M 15 1787 141 M T D A L H M LT D 15 1788 148 L T D L S A I I L T 15 1789 152 S A I I L T L L A L 151790 168 P T K R F T F G F H 15 1791 170 K R F T F G F H R L 15 1792 172F T F G F H R L E V 15 1793 187 S V L L V Y I L M G 15 1794 193 I L M GF L L Y E A 15 1795 282 L V Q S V G V L I A 15 1796 289 L I A A Y I I RF K 15 1797 315 L V A F T T F R I I 15 1798 342 D Y I K E A L M K I 151799 407 T I Q L Q S Y R Q E 15 1800 HLA-A3 decamers 157 T L L A L W L SS K 29 1801  91 K V D S C D N C S K 25 1802 293 Y I I R F K P E Y K 251803  82 P L T N S Q L S L K 24 1804 102 R E I L K Q R K V K 23 1805 202A V Q R T I H M N Y 23 1806 268 S L A V R A A F V H 23 1807 341 V D Y IK E A L M K 22 1808 278 A L G D L V Q S V G 21 1809 289 L I A A Y I I RF K 21 1810 328 V V I I L E G V P S 21 1811 377 Q L I P G S S S K W 211812 395 H L L L N T F G M Y 21 1813 396 L L L N T F G M Y R 21 1814  13M L R K D D A P L F 20 1815 137 S L A I M T D A L H 20 1816 234 F L L NQ S G H R H 20 1817 334 G V P S H L N V D Y 20 1818 376 I Q L I P G S SS K 20 1819  20 P L F L N D T S A F 19 1820  45 K L R V V V A D D G 191821 154 I I L T L L A L W L 19 1822 180 E V L S A M I S V L 19 1823 198L L Y E A V Q R T I 19 1824 216 D I M L I T A A V G 19 1825 270 A V R AA F V H A L 19 1826 275 F V H A L G D L V Q 19 1827 288 V L I A A Y I IR F 19 1828 322 R I I W D T V V I I 19 1829  22 F L N D T S A F D F 181830  48 V V V A D D G S E A 18 1831  76 L L D Q D L P L T N 18 1832 104I L K Q R K V K A R 18 1833 112 A R L T I A A V L Y 18 1834 113 R L T IA A V L Y L 18 1835 115 T I A A V L Y L L F 18 1836 144 A L H M L T D LS A 18 1837 153 A I I L T L L A L W 18 1838 178 R L E V L S A M I S 181839 187 S V L L V Y I L M G 18 1840 190 L V Y I L M G F L L 18 1841 218M L I T A A V G V A 18 1842 219 L I T A A V G V A V 18 1843 313 S L L VA F T T F R 18 1844 329 V I I L E G V P S H 18 1845  7 W K R L K S M L RK 17 1846  47 R V V V A D D G S E 17 1847 100 K Q R E I L K Q R K 171848 109 K V K A R L T I A A 17 1849 111 K A R L T I A A V L 17 1850 122L L F M I G E L V G 17 1851 129 L V G G Y I A N S L 17 1852 160 A L W LS S K S P T 17 1853 188 V L L V Y I L M G F 17 1854 196 G F L L Y E A VQ R 17 1855 223 A V G V A V N V I M 17 1856 281 D L V Q S V G V L I 171857 285 S V G V L I A A Y I 17 1858 287 G V L I A A Y I I R 17 1859 330I I L E G V P S H L 17 1860 347 A L M K I E D V Y S 17 1861 353 D V Y SV E D L N I 17 1862 375 H I Q L I P G S S S 17 1863  9 R L K S M L R K DD 16 1864  39 G L S R F N K L R V 16 1865  87 Q L S L K V D S C D 161866 118 A V L Y L L F M I G 16 1867 161 L W L S S K S P T K 16 1868 162W L S S K S P T K R 16 1869 181 V L S A M I S V L L 16 1870 225 G V A VN V I M G F 16 1871 227 A V N V I M G F L L 16 1872 229 N V I M G F L LN Q 16 1873 327 T V V I I L E G V P 16 1874 350 K I E D V Y S V E D 161875 360 L N I W S L T S G K 16 1876 409 Q L Q S Y R Q E V D 16 1877 416E V D R T C A N C Q 16 1878  2 A G S G A W K R L K 15 1879  33 D E A G DE G L S R 15 1880  68 T L Q A D D D S L L 15 1881 119 V L Y L L F M I GE 15 1882 121 Y L L F M I G E L V 15 1883 128 E L V G G Y I A N S 151884 158 L L A L W L S S K S 15 1885 183 S A M I S V L L V Y 15 1886 301Y K I A D P I C T Y 15 1887  61 P V N G A H P T L Q 14 1888  75 S L L DQ D L P L T 14 1889 103 E I L K Q R K V K A 14 1890 133 Y I A N S L A IM T 14 1891 147 M L T D L S A I I L 14 1892 193 I L M G F L L Y E A 141893 284 Q S V G V L I A A Y 14 1894 302 K I A D P I C T Y V 14 1895 331I L E G V P S H L N 14 1896 356 S V E D L N I W S L 14 1897 359 D L N IW S L T S G 14 1898 383 S S K W E E V Q S K 14 1899 HLA-B*0702 decamers 60 R P V N G A H P T L 22 1900  81 L P L T N S Q L S L 22 1901 305 D PI C T Y V F S L 21 1902  19 A P L F L N D T S A 19 1903 335 V P S H L NV D Y I 19 1904 167 S P T K R F T F G F 18 1905 298 K P E Y K I A D P I18 1906 270 A V R A A F V H A L 17 1907 111 K A R L T I A A V L 15 1908181 V L S A M I S V L L 15 1909 389 V Q S K A N H L L L 15 1910 113 R LT I A A V L Y L 14 1911 150 D L S A I I L T L L 14 1912  14 L R K D D AP L F L 13 1913  57 A P E R P V N G A H 13 1914  74 D S L L D Q D L P L13 1915 129 L V G G Y I A N S L 13 1916 136 N S L A I M T D A L 13 1917139 A I M T D A L H M L 13 1918 152 S A I I L T L L A L 13 1919 154 I IL T L L A L W L 13 1920 185 M I S V L L V Y I L 13 1921 249 L P S N S PT R G S 13 1922 351 I E D V Y S V E D L 13 1923  12 S M L R K D D A P L12 1924  37 D E G L S R F N K L 12 1925  42 R F N K L R V V V A 12 1926142 T D A L H M L T D L 12 1927 149 T D L S A I I L T L 12 1928 170 K RF T F G F H R L 12 1929 173 T F G F H R L E V L 12 1930 180 E V L S A MI S V L 12 1931 222 A A V G V A V N V I 12 1932 227 A V N V I M G F L L12 1933 240 G H R H S H S H S L 12 1934 260 C E R N H G Q D S L 12 1935262 R N H G Q D S L A V 12 1936 273 A A F V H A L G D L 12 1937 280 G DL V Q S V G V L 12 1938 323 I I W D T V V I I L 12 1939 365 L T S G K ST A I V 12 1940 369 K S T A I V H I Q L 12 1941 379 I P G S S S K W E E12 1942 401 F G M Y R C T I Q L 12 1943  1 M A G S G A W K R L 11 1944 5 G A W K R L K S M L 11 1945  31 F S D E A G D E G L 11 1946  62 V N GA H P T L Q A 11 1947  68 T L Q A D D D S L L 11 1948  72 D D D S L L DQ D L 11 1949  79 Q D L P L T N S Q L 11 1950  96 D N C S K Q R E I L 111951 105 L K Q R K V K A R L 11 1952 114 L T I A A V L Y L L 11 1953 116I A A V L Y L L F M 11 1954 135 A N S L A I M T D A 11 1955 172 F T F GF H R L E V 11 1956 189 L L V Y I L M G F L 11 1957 212 E I N G D I M LI T 11 1958 223 A V G V A V N V I M 11 1959 226 V A V N V I M G F L 111960 266 Q D S L A V R A A F 11 1961 303 I A D P I C T Y V F 11 1962 306P I C T Y V F S L L 11 1963 330 I I L E G V P S H L 11 1964 339 L N V DY I K E A L 11 1965 387 E E V Q S K A N H L 11 1966 388 E V Q S K A N HL L 11 1967 MHC Class 1 nonamer and decamer analysis of 108P5H8 flankingthe D to E mutation at amino acid 30. Listed are scores that fall withinthe top 50% (rounded up) of all scores for a selected allele of the108P5H8 variant 1 sequence that does not contain the mutation. SEQ. ID.Pos 1 2 3 4 5 6 7 8 9 score No. HLA-A*0201 nonamers  27 S A F E F S D EA 16 2082  22 F L N D T S A F E 15 2083 HLA-A*0203 nonamers  27 S A F EF S D E A 11 2084 HLA-A26 nonamers  25 D T S A F E F S D 17 2085HLA-B*1510 nonamers  23 L N D T S A F E F 7 2086 HLA-B*5101 decamers  27S A F E F S D E A 13 2087 SEQ. ID. Pos 1 2 3 4 5 6 7 8 9 0 score No.HLA-A*0201 decamers  22 F L N D T S A F E F 15 2088 HLA-A1 decamers  23L N D T S A F E F S 13 2089 HLA-A26 decamers  25 D T S A F E F S D E 212090  22 F L N D T S A F E F 20 2091 HLA-A3 decamers  22 F L N D T S A FE F 18 2092

[0851] TABLE XXIII MHC Class II 15-mer analysis of 108P5H8 for selectedalleles. Listed are scores that fall within the top 50% (rounded up) ofall scores for the selected allele. SEQ. ID Pos 1 2 3 4 5 6 7 8 9 0 1 23 4 5 score No. HLA-DRB1*0101 15-mers 326 D T V V I I L E G V P S H L N36 2093 188 V L L V Y I L M G F L L Y E A 35 2094 145 L H M L T D L S AI I L T L L 33 2095 123 L F M I G E L V G G Y I A N S 32 2096 152 S A II L T L L A L W L S S K 32 2097 176 F H R L E V L S A M I S V L L 322098 283 V Q S V G V L I A A Y I I R F 32 2099 119 V L Y L L F M I G E LV G G Y 31 2100 225 G V A V N V I M G F L L N Q S 31 2101 359 D L N I WS L T S G K S T A I 31 2102 373 I V H I Q L I P G S S S K W E 31 2103318 F T T F R I I W D T V V I I L 30 2104  40 L S R F N K L R V V V A DD G 29 2105 215 G D I M L I T A A V G V A V N 29 2106  11 K S M L R K DD A P L F L N D 28 2107 179 L E V L S A M I S V L L V Y I 28 2108 173 TF G F H R L E V L S A M I S 27 2109 272 R A A F V H A L G D L V Q S V 272110 309 T Y V F S L L V A F T T F R I 27 2111 371 T A I V H I Q L I P GS S S K 27 2112 153 A I I L T L L A L W L S S K S 26 2113 325 W D T V VI I L E G V P S H L 26 2114  4 S G A W K R L K S M L R K D D 25 2115  20P L F L N D T S A F D F S D E 25 2116  46 L R V V V A D D G S E A P E R25 2117 101 Q R E I L K Q R K V K A R L T 25 2118 142 T D A L H M L T DL S A I I L 25 2119 192 Y I L M G F L L Y E A V Q R T 25 2120 217 I M LI T A A V G V A V N V I 25 2121 279 L G D L V Q S V G V L I A A Y 252122 345 K E A L M K I E D V Y S V E D 25 2123 392 K A N H L L L N T F GM Y R C 25 2124  43 F N K L R V V V A D D G S E A 24 2125  56 E A P E RP V N G A H P T L Q 24 2126 124 F M I G E L V G G Y I A N S L 24 2127127 G E L V G G Y I A N S L A I M 24 2128 156 L T L L A L W L S S K S PT K 24 2129 183 S A M I S V L L V Y I L M G F 24 2130 284 Q S V G V L IA A Y I I R F K 24 2131 354 V Y S V E D L N I W S L T S G 24 2132 362 IW S L T S G K S T A I V H I 24 2133  18 D A P L F L N D T S A F D F S 232134  47 R V V V A D D G S E A P E R P 23 2135  78 D Q D L P L T N S Q LS L K V 23 2136 107 Q R K V K A R L T I A A V L Y 23 2137 148 L T D L SA I I L T L L A L W 23 2138 149 T D L S A I I L T L L A L W L 23 2139157 T L L A L W L S S K S P T K R 23 2140 187 S V L L V Y I L M G F L LY E 23 2141 213 I N G D I M L I T A A V G V A 23 2142 214 N G D I M L IT A A V G V A V 23 2143 277 H A L G D L V Q S V G V L I A 23 2144 280 GD L V Q S V G V L I A A Y I 23 2145 312 F S L L V A F T T F R I I W D 232146 351 I E D V Y S V E D L N I W S L 23 2147 383 S S K W E E V Q S K AN H L L 23 2148  81 L P L T N S Q L S L K V D S C 22 2149 113 R L T I AA V L Y L L F M I G 22 2150 144 A L H M L T D L S A I I L T L 22 2151182 L S A M I S V L L V Y I L M G 22 2152 207 I H M N Y E I N G D I M LI T 22 2153 208 H M N Y E I N G D I M L I T A 22 2154 232 M G F L L N QS G H R H S H S 22 2155 243 H S H S H S L P S N S P T R G 22 2156 265 GQ D S L A V R A A F V H A L 22 2157 328 V V I I L E G V P S H L N V D 222158 338 H L N V D Y I K E A L M K I E 22 2159 372 A I V H I Q L I P G SS S K W 22 2160  99 S K Q R E I L K Q R K V K A R 21 2161 305 D P I C TY V F S L L V A F T 21 2162 340 N V D Y I K E A L M K I E D V 21 2163111 K A R L T I A A V L Y L L F M 20 2164 134 I A N S L A I M T D A L HM L 20 2165 141 M T D A L H M L T D L S A I I 20 2166 175 G F H R L E VL S A M I S V L 20 2167 194 L M G F L L Y E A V Q R T I H 20 2168 198 LL Y E A V Q R T I H M N Y E 20 2169 260 C E R N H G Q D S L A V R A A 202170 292 A Y I I R F K P E Y K I A D P 20 2171 298 K P E Y K I A D P I CT Y V F 20 2172 301 Y K I A D P I C T Y V F S L L 20 2173 307 I C T Y VF S L L V A F T T F 20 2174 329 V I I L E G V P S H L N V D Y 20 2175346 E A L M K I E D V Y S V E D L 20 2176 348 L M K I E D V Y S V E D LN I 20 2177 356 S V E D L N I W S L T S G K S 20 2178 360 L N I W S L TS G K S T A I V 20 2179 401 F G M Y R C T I Q L Q S Y R Q 20 2180 104 IL K Q R K V K A R L T I A A 19 2181 109 K V K A R L T I A A V L Y L L 192182 117 A A V L Y L L F M I G E L V G 19 2183 118 A V L Y L L F M I G EL V G G 19 2184 150 D L S A I I L T L L A L W L S 19 2185 165 S K S P TK R F T F G F H R L 19 2186 171 R F T F G F H R L E V L S A M 19 2187233 G F L L N Q S G H R H S H S H 19 2188  17 D D A P L F L N D T S A FD F 18 2189  28 A F D F S D E A G D E G L S R 18 2190 130 V G G Y I A NS L A I M T D A 18 2191 184 A M I S V L L V Y I L M G F L 18 2192 186 IS V L L V Y I L M G F L L Y 18 2193 189 L L V Y I L M G F L L Y E A V 182194 212 E I N G D I M L I T A A V G V 18 2195 223 A V G V A V N V I M GF L L N 18 2196 268 S L A V R A A F V H A L G D L 18 2197 313 S L L V AF T T F R I I W D T 18 2198 336 P S H L N V D Y I K E A L M K 18 2199HLA-DRB1*0301 (DR17) 15-mers  11 K S M L R K D D A P L F L N D 30 2200 66 H P T L Q A D D D S L L D Q D 29 2201 137 S L A I M T D A L H M L TD L 28 2202 332 L E G V P S H L N V D Y I K E 27 2203 386 W E E V Q S KA N H L L L N T 26 2204 144 A L H M L T D L S A I I L T L 24 2205  87 QL S L K V D S C D N C S K Q 23 2206  47 R V V V A D D G S E A P E R P 222207  72 D D D S L L D Q D L P L T N S 22 2208  74 D S L L D Q D L P L TN S Q L 22 2209 152 S A I I L T L L A L W L S S K 22 2210 320 T F R I IW D T V V I I L E G 22 2211  10 L K S M L R K D D A P L F L N 21 2212 19 A P L F L N D T S A F D F S D 21 2213  46 L R V V V A D D G S E A PE R 21 2214 113 R L T I A A V L Y L L F M I G 21 2215 178 R L E V L S AM I S V L L V Y 21 2216 210 N Y E I N G D I M L I T A A V 21 2217 328 VV I I L E G V P S H L N V D 21 2218 393 A N H L L L N T F G M Y R C T 212219  12 S M L R K D D A P L F L N D T 20 2220  18 D A P L F L N D T S AF D F S 20 2221 101 Q R E I L K Q R K V K A R L T 20 2222 111 K A R L TI A A V L Y L L F M 20 2223 122 L L F M I G E L V G G Y I A N 20 2224145 L H M L T D L S A I I L T L L 20 2225 179 L E V L S A M I S V L L VY I 20 2226 186 I S V L L V Y I L M G F L L Y 20 2227 187 S V L L V Y IL M G F L L Y E 20 2228 268 S L A V R A A F V H A L G D L 20 2229 299 PE Y K I A D P I C T Y V F S 20 2230 336 P S H L N V D Y I K E A L M K 202231  7 W K R L K S M L R K D D A P L 19 2232  20 P L F L N D T S A F DF S D E 19 2233 120 L Y L L F M I G E L V G G Y I 19 2234 127 G E L V GG Y I A N S L A I M 19 2235 148 L T D L S A I I L T L L A L W 19 2236183 S A M I S V L L V Y I L M G F 19 2237 188 V L L V Y I L M G F L L YE A 19 2238 200 Y E A V Q R T I H M N Y E I N 19 2239 225 G V A V N V IM G F L L N Q S 19 2240 286 V G V L I A A Y I I R F K P E 19 2241 292 AY I I R F K P E Y K I A D P 19 2242 304 A D P I C T Y V F S L L V A F 192243 338 H L N V D Y I K E A L M K I E 19 2244 353 D V Y S V E D L N I WS L T S 19 2245  79 Q D L P L T N S Q L S L K V D 18 2246 158 L L A L WL S S K S P T K R F 18 2247 204 Q R T I H M N Y E I N G D I M 18 2248223 A V G V A V N V I M G F L L N 18 2249 229 N V I M G F L L N Q S G HR H 18 2250 321 F R I I W D T V V I I L E G V 18 2251 325 W D T V V I IL E G V P S H L 18 2252 354 V Y S V E D L N I W S L T S G 18 2253  37 DE G L S R F N K L R V V V A 17 2254  77 L D Q D L P L T N S Q L S L K 172255  89 S L K V D S C D N C S K Q R E 17 2256 196 G F L L Y E A V Q R TI H M N 17 2257 221 T A A V G V A V N V I M G F L 17 2258 264 H G Q D SL A V R A A F V H A 17 2259 294 I I R F K P E Y K I A D P I C 17 2260344 I K E A L M K I E D V Y S V E 17 2261 407 T I Q L Q S Y R Q E V D RT C 17 2262 163 L S S K S P T K R F T F G F H 16 2263 171 R F T F G F HR L E V L S A M 16 2264 206 T I H M N Y E I N G D I M L I 16 2265 258 SG C E R N H G Q D S L A V R 16 2266 276 V H A L G D L V Q S V G V L I 162267 337 S H L N V D Y I K E A L M K I 16 2268  3 G S G A W K R L K S ML R K D 15 2269  26 T S A F D F S D E A G D E G L 15 2270  94 S C D N CS K Q R E I L K Q R 15 2271 103 E I L K Q R K V K A R L T I A 15 2272151 L S A I I L T L L A L W L S S 15 2273 290 I A A Y I I R F K P E Y KI A 15 2274 HLA-DRB1*0401 (DR4Dw4) 15-mers 383 S S K W E E V Q S K A N HL L 28 2275 138 L A I M T D A L H M L T D L S 26 2276 144 A L H M L T DL S A I I L T L 26 2277 196 G F L L Y E A V Q R T I H M N 26 2278 206 TI H M N Y E I N G D I M L I 26 2279 221 T A A V G V A V N V I M G F L 262280 312 F S L L V A F T T F R I I W D 26 2281 329 V I I L E G V P S H LN V D Y 26 2282 359 D L N I W S L T S G K S T A I 26 2283  4 S G A W K RL K S M L R K D D 22 2284  19 A P L F L N D T S A F D F S D 22 2285  40L S R F N K L R V V V A D D G 22 2286 118 A V L Y L L F M I G E L V G G22 2287 159 L A L W L S S K S P T K R F T 22 2288 173 T F G F H R L E VL S A M I S 22 2289 197 F L L Y E A V Q R T I H M N Y 22 2290 272 R A AF V H A L G D L V Q S V 22 2291 298 K P E Y K I A D P I C T Y V F 222292 309 T Y V F S L L V A F T T F R I 22 2293 318 F T T F R I I W D T VV I I L 22 2294 340 N V D Y I K E A L M K I E D V 22 2295 401 F G M Y RC T I Q L Q S Y R Q 22 2296  37 D E G L S R F N K L R V V V A 20 2297 46 L R V V V A D D G S E A P E R 20 2298  47 R V V V A D D G S E A P ER P 20 2299  59 E R P V N G A H P T L Q A D D 20 2300  66 H P T L Q A DD D S L L D Q D 20 2301  74 D S L L D Q D L P L T N S Q L 20 2302  78 DQ D L P L T N S Q L S L K V 20 2303  89 S L K V D S C D N C S K Q R E 202304 111 K A R L T I A A V L Y L L F M 20 2305 123 L F M I G E L V G G YI A N S 20 2306 137 S L A I M T D A L H M L T D L 20 2307 142 T D A L HM L T D L S A I I L 20 2308 145 L H M L T D L S A I I L T L L 20 2309148 L T D L S A I I L T L L A L W 20 2310 152 S A I I L T L L A L W L SS K 20 2311 153 A I I L T L L A L W L S S K S 20 2312 155 I L T L L A LW L S S K S P T 20 2313 156 L T L L A L W L S S K S P T K 20 2314 176 FH R L E V L S A M I S V L L 20 2315 178 R L E V L S A M I S V L L V Y 202316 179 L E V L S A M I S V L L V Y I 20 2317 182 L S A M I S V L L V YI L M G 20 2318 183 S A M I S V L L V Y I L M G F 20 2319 187 S V L L VY I L M G F L L Y E 20 2320 188 V L L V Y I L M G F L L Y E A 20 2321191 V Y I L M G F L L Y E A V Q R 20 2322 192 Y I L M G F L L Y E A V QR T 20 2323 195 M G F L L Y E A V Q R T I H M 20 2324 210 N Y E I N G DI M L I T A A V 20 2325 216 D I M L I T A A V G V A V N V 20 2326 217 IM L I T A A V G V A V N V I 20 2327 225 G V A V N V I M G F L L N Q S 202328 228 V N V I M G F L L N Q S G H R 20 2329 229 N V I M G F L L N Q SG H R H 20 2330 233 G F L L N Q S G H R H S H S H 20 2331 268 S L A V RA A F V H A L G D L 20 2332 273 A A F V H A L G D L V Q S V G 20 2333276 V H A L G D L V Q S V G V L I 20 2334 280 G D L V Q S V G V L I A AY I 20 2335 283 V Q S V G V L I A A Y I I R F 20 2336 291 A A Y I I R FK P E Y K I A D 20 2337 308 C T Y V F S L L V A F T T F R 20 2338 311 VF S L L V A F T T F R I I W 20 2339 320 T F R I I W D T V V I I L E G 202340 326 D T V V I I L E G V P S H L N 20 2341 336 P S H L N V D Y I K EA L M K 20 2342 338 H L N V D Y I K E A L M K I E 20 2343 345 K E A L MK I E D V Y S V E D 20 2344 346 E A L M K I E D V Y S V E D L 20 2345348 L M K I E D V Y S V E D L N I 20 2346 351 I E D V Y S V E D L N I WS L 20 2347 354 V Y S V E D L N I W S L T S G 20 2348 362 I W S L T S GK S T A I V H I 20 2349 373 I V H I Q L I P G S S S K W E 20 2350  16 KD D A P L F L N D T S A F D 18 2351  24 N D T S A F D F S D E A G D E 182352  34 E A G D E G L S R F N K L R V 18 2353  77 L D Q D L P L T N S QL S L K 18 2354  86 S Q L S L K V D S C D N C S K 18 2355  99 S K Q R EI L K Q R K V K A R 18 2356 128 E L V G G Y I A N S L A I M T 18 2357129 L V G G Y I A N S L A I M T D 18 2358 134 I A N S L A I M T D A L HM L 18 2359 149 T D L S A I I L T L L A L W L 18 2360 170 K R F T F G FH R L E V L S A 18 2361 175 G F H R L E V L S A M I S V L 18 2362 213 IN G D I M L I T A A V G V A 18 2363 255 T R G S G C E R N H G Q D S L 182364 277 H A L G D L V Q S V G V L I A 18 2365 305 D P I C T Y V F S L LV A F T 18 2366 353 D V Y S V E D L N I W S L T S 18 2367 356 S V E D LN I W S L T S G K S 18 2368 367 S G K S T A I V H I Q L I P G 18 2369382 S S S K W E E V Q S K A N H L 18 2370 387 E E V Q S K A N H L L L NT F 18 2371 392 K A N H L L L N T F G M Y R C 18 2372 404 Y R C T I Q LQ S Y R Q E V D 18 2373 411 Q S Y R Q E V D R T C A N C Q 18 2374 412 SY R Q E V D R T C A N C Q S 18 2375  28 A F D F S D E A G D E G L S R 162376 130 V G G Y I A N S L A I M T D A 16 2377 169 T K R F T F G F H R LE V L S 16 2378 171 R F T F G F H R L E V L S A M 16 2379 208 H M N Y EI N G D I M L I T A 16 2380 231 I M G F L L N Q S G H R H S H 16 2381294 I I R F K P E Y K I A D P I C 16 2382 315 L V A F T T F R I I W D TV V 16 2383 322 R I I W D T V V I I L E G V P 16 2384 352 E D V Y S V ED L N I W S L T 16 2385 360 L N I W S L T S G K S T A I V 16 2386 398 LN T F G M Y R C T I Q L Q S 16 2387 410 L Q S Y R Q E V D R T C A N C 162388 107 Q R K V K A R L T I A A V L Y 15 2389 386 W E E V Q S K A N H LL L N T 15 2390  7 W K R L K S M L R K D D A P L 14 2391  11 K S M L R KD D A P L F L N D 14 2392  18 D A P L F L N D T S A F D F S 14 2393  43F N K L R V V V A D D G S E A 14 2394  45 K L R V V V A D D G S E A P E14 2395  73 D D S L L D Q D L P L T N S Q 14 2396  80 D L P L T N S Q LS L K V D S 14 2397  87 Q L S L K V D S C D N C S K Q 14 2398 101 Q R EI L K Q R K V K A R L T 14 2399 113 R L T I A A V L Y L L F M I G 142400 116 I A A V L Y L L F M I G E L V 14 2401 117 A A V L Y L L F M I GE L V G 14 2402 119 V L Y L L F M I G E L V G G Y 14 2403 120 L Y L L FM I G E L V G G Y I 14 2404 122 L L F M I G E L V G G Y I A N 14 2405126 I G E L V G G Y I A N S L A I 14 2406 127 G E L V G G Y I A N S L AI M 14 2407 131 G G Y I A N S L A I M T D A L 14 2408 158 L L A L W L SS K S P T K R F 14 2409 185 M I S V L L V Y I L M G F L L 14 2410 200 YE A V Q R T I H M N Y E I N 14 2411 214 N G D I M L I T A A V G V A V 142412 215 G D I M L I T A A V G V A V N 14 2413 227 A V N V I M G F L L NQ S G H 14 2414 279 L G D L V Q S V G V L I A A Y 14 2415 285 S V G V LI A A Y I I R F K P 14 2416 286 V G V L I A A Y I I R F K P E 14 2417287 G V L I A A Y I I R F K P E Y 14 2418 300 E Y K I A D P I C T Y V FS L 14 2419 304 A D P I C T Y V F S L L V A F 14 2420 313 S L L V A F TT F R I I W D T 14 2421 321 F R I I W D T V V I I L E G V 14 2422 325 WD T V V I I L E G V P S H L 14 2423 327 T V V I I L E G V P S H L N V 142424 328 V V I I L E G V P S H L N V D 14 2425 332 L E G V P S H L N V DY I K E 14 2426 341 V D Y I K E A L M K I E D V Y 14 2427 370 S T A I VH I Q L I P G S S S 14 2428 371 T A I V H I Q L I P G S S S K 14 2429375 H I Q L I P G S S S K W E E V 14 2430 376 I Q L I P G S S S K W E EV Q 14 2431 395 H L L L N T F G M Y R C T I Q 14 2432 400 T F G M Y R CT I Q L Q S Y R 14 2433 407 T I Q L Q S Y R Q E V D R T C 14 2434 414 RQ E V D R T C A N C Q S S S 14 2435 HLA-DRB1*1101 15-mers 197 F L L Y EA V Q R T I H M N Y 25 2436  40 L S R F N K L R V V V A D D G 23 2437169 T K R F T F G F H R L E V L S 22 2438 173 T F G F H R L E V L S A MI S 22 2439  43 F N K L R V V V A D D G S E A 21 2440 101 Q R E I L K QR K V K A R L T 21 2441 153 A I I L T L L A L W L S S K S 21 2442 233 GF L L N Q S G H R H S H S H 21 2443 276 V H A L G D L V Q S V G V L I 212444 288 V L I A A Y I I R F K P E Y K 21 2445  7 W K R L K S M L R K DD A P L 20 2446  8 K R L K S M L R K D D A P L F 20 2447 120 L Y L L F MI G E L V G G Y I 20 2448 176 F H R L E V L S A M I S V L L 20 2449 411Q S Y R Q E V D R T C A N C Q 20 2450 116 I A A V L Y L L F M I G E L V19 2451 214 N G D I M L I T A A V G V A V 19 2452 280 G D L V Q S V G VL I A A Y I 19 2453 322 R I I W D T V V I I L E G V P 19 2454 325 W D TV V I I L E G V P S H L 19 2455 359 D L N I W S L T S G K S T A I 192456 142 T D A L H M L T D L S A I I L 18 2457 185 M I S V L L V Y I L MG F L L 18 2458 229 N V I M G F L L N Q S G H R H 18 2459 290 I A A Y II R F K P E Y K I A 18 2460 294 I I R F K P E Y K I A D P I C 18 2461309 T Y V F S L L V A F T T F R I 18 2462 326 D T V V I I L E G V P S HL N 18 2463 345 K E A L M K I E D V Y S V E D 18 2464 367 S G K S T A IV H I Q L I P G 18 2465 370 S T A I V H I Q L I P G S S S 18 2466 373 IV H I Q L I P G S S S K W E 18 2467  4 S G A W K R L K S M L R K D D 172468 138 L A I M T D A L H M L T D L S 17 2469 189 L L V Y I L M G F L LY E A V 17 2470 269 L A V R A A F V H A L G D L V 17 2471 318 F T T F RI I W D T V V I I L 17 2472  28 A F D F S D E A G D E G L S R 16 2473 37 D E G L S R F N K L R V V V A 16 2474  98 C S K Q R E I L K Q R K VK A 16 2475 121 Y L L F M I G E L V G G Y I A 16 2476 383 S S K W E E VQ S K A N H L L 16 2477 401 F G M Y R C T I Q L Q S Y R Q 16 2478  1 M AG S G A W K R L K S M L R 15 2479 145 L H M L T D L S A I I L T L L 152480 239 S G H R H S H S H S L P S N S 15 2481 323 I I W D T V V I I L EG V P S 15 2482 397 L L N T F G M Y R C T I Q L Q 15 2483  34 E A G D EG L S R F N K L R V 14 2484  83 L T N S Q L S L K V D S C D N 14 2485123 L F M I G E L V G G Y I A N S 14 2486 158 L L A L W L S S K S P T KR F 14 2487 183 S A M I S V L L V Y I L M G F 14 2488 200 Y E A V Q R TI H M N Y E I N 14 2489 225 G V A V N V I M G F L L N Q S 14 2490 235 LL N Q S G H R H S H S H S L 14 2491 237 N Q S G H R H S H S H S L P S 142492 246 S H S L P S N S P T R G S G C 14 2493 248 S L P S N S P T R G SG C E R 14 2494 254 P T R G S G C E R N H G Q D S 14 2495 342 D Y I K EA L M K I E D V Y S 14 2496 361 N I W S L T S G K S T A I V H 14 2497 44 N K L R V V V A D D G S E A P 13 2498  71 A D D D S L L D Q D L P LT N 13 2499  78 D Q D L P L T N S Q L S L K V 13 2500 113 R L T I A A VL Y L L F M I G 13 2501 119 V L Y L L F M I G E L V G G Y 13 2502 124 FM I G E L V G G Y I A N S L 13 2503 128 E L V G G Y I A N S L A I M T 132504 135 A N S L A I M T D A L H M L T 13 2505 141 M T D A L H M L T D LS A I I 13 2506 148 L T D L S A I I L T L L A L W 13 2507 149 T D L S AI I L T L L A L W L 13 2508 156 L T L L A L W L S S K S P T K 13 2509179 L E V L S A M I S V L L V Y I 13 2510 188 V L L V Y I L M G F L L YE A 13 2511 216 D I M L I T A A V G V A V N V 13 2512 263 N H G Q D S LA V R A A F V H 13 2513 273 A A F V H A L G D L V Q S V G 13 2514 283 VQ S V G V L I A A Y I I R F 13 2515 308 C T Y V F S L L V A F T T F R 132516 329 V I I L E G V P S H L N V D Y 13 2517 336 P S H L N V D Y I K EA L M K 13 2518 338 H L N V D Y I K E A L M K I E 13 2519 410 L Q S Y RQ E V D R T C A N C 13 2520  17 D D A P L F L N D T S A F D F 12 2521 46 L R V V V A D D G S E A P E R 12 2522  47 R V V V A D D G S E A P ER P 12 2523  56 E A P E R P V N G A H P T L Q 12 2524  75 S L L D Q D LP L T N S Q L S 12 2525 103 E I L K Q R K V K A R L T I A 12 2526 107 QR K V K A R L T I A A V L Y 12 2527 117 A A V L Y L L F M I G E L V G 122528 126 I G E L V G G Y I A N S L A I 12 2529 152 S A I I L T L L A L WL S S K 12 2530 155 I L T L L A L W L S S K S P T 12 2531 157 T L L A LW L S S K S P T K R 12 2532 182 L S A M I S V L L V Y I L M G 12 2533187 S V L L V Y I L M G F L L Y E 12 2534 191 V Y I L M G F L L Y E A VQ R 12 2535 192 Y I L M G F L L Y E A V Q R T 12 2536 204 Q R T I H M NY E I N G D I M 12 2537 211 Y E I N G D I M L I T A A V G 12 2538 212 EI N G D I M L I T A A V G V 12 2539 222 A A V G V A V N V I M G F L L 122540 228 V N V I M G F L L N Q S G H R 12 2541 243 H S H S H S L P S N SP T R G 12 2542 315 L V A F T T F R I I W D T V V 12 2543 348 L M K I ED V Y S V E D L N I 12 2544 351 I E D V Y S V E D L N I W S L 12 2545352 E D V Y S V E D L N I W S L T 12 2546 354 V Y S V E D L N I W S L TS G 12 2547 356 S V E D L N I W S L T S G K S 12 2548 357 V E D L N I WS L T S G K S T 12 2549 371 T A I V H I Q L I P G S S S K 12 2550 372 AI V H I Q L I P G S S S K W 12 2551 391 S K A N H L L L N T F G M Y R 122552

[0852] MHC class II analysis of 108P5H8 flanking the D to E mutation atamino acid 30. Listed are scores that fall within the top 50% (roundedup) of all scores for a selected allele of the 108P5H8 variant 1sequence that does not contain the mutation. SEQ. ID Pos 1 2 3 4 5 6 7 89 0 1 2 3 4 5 score No. HLA-DRB1*0101 15-mers 20 P L F L N D T S A F E FS D E 25 2553 18 D A P L F L N D T S A F E F S 23 2554 17 D D A P L F LN D T S A F E F 18 2555 28 A F E F S D E A G D E G L S R 18 2556HLA-DRB1*0301 (DR17) 15-mers 19 A P L F L N D T S A F E F S D 21 2557 18D A P L F L N D T S A F E F S 20 2558 20 P L F L N D T S A F E F S D E19 2559 26 T S A F E F S D E A G D E G L 15 2560 HLA-DRB1*0401 (DR4Dw4)15-mers 19 A P L F L N D T S A F E F S D 22 2561 16 K D D A P L F L N DT S A F E 18 2562 24 N D T S A F E F S D E A G D E 18 2563 28 A F E F SD E A G D E G L S R 16 2564 18 D A P L F L N D T S A F E F S 14 2565HLA-DRB1*1101 15-mers 28 A F E F S D E A G D E G L S R 16 2566 17 D D AP L F L N D T S A F E F 12 2567

1. A composition comprising: a substance that modulates the status of108P5H8, or a molecule that is modulated by 108P5H8 whereby the statusof a cell that expresses 108P5H8 is modulated.
 2. The composition ofclaim 1, further comprising a pharmaceutically acceptable carrier.
 3. Apharmaceutical composition that comprises the composition of claim 1 ina human unit dose form.
 4. A composition of claim 1 wherein thesubstance comprises an antibody or fragment thereof that specificallybinds to a 108P5H8-related protein.
 5. The antibody or fragment thereofof claim 4, which is monoclonal.
 6. A recombinant protein comprising anantigen-binding region of a monoclonal antibody of claim
 5. 7. Theantibody or fragment thereof of claim 4, which is labeled with adetectable marker.
 8. The recombinant protein of claim 6, which islabeled with a detectable marker.
 9. The antibody fragment of anantibody of claim 4, which is an Fab, F(ab′)2, Fv or sFv fragment. 10.The antibody of claim 4, which is a human antibody, a humanized antibodyor a chimeric antibody.
 11. A non-human transgenic animal that producesan antibody of claim
 4. 12. A hybridoma that produces an antibody ofclaim
 5. 13. A single chain monoclonal antibody that immunospecificallybinds to a 108P5H8-related protein, and that comprises the variabledomains of the heavy and light chains of a monoclonal antibody of claim5.
 14. A vector comprising a polynucleotide that encodes a single chainmonoclonal antibody of claim
 13. 15. A method of delivering a cytotoxicagent or a diagnostic agent to a cell that expresses 108P5H8, saidmethod comprising: providing the cytotoxic agent or the diagnostic agentconjugated to an antibody or fragment thereof of claim 4; and, exposingthe cell to the antibody-agent or fragment-agent conjugate.
 16. Acomposition of claim 1 wherein the substance comprises a polynucleotidethat encodes an antibody or fragment thereof either of whichimmunospecifically binds to an 108P5H8-related protein.
 17. Acomposition of claim 3 wherein the substance comprises a 108P5H8-relatedprotein.
 18. The composition of claim 17, further comprising antigenpresenting cells.
 19. The composition of claim 1 wherein the substancecomprises an analog of a peptide of eight, nine, ten, or elevencontiguous amino acids of FIG. 2 (SEQ ID Nos.: 2570, 2572, 2574).
 20. Acomposition of claim 1 wherein the substance comprises a CTL polypeptideepitope of the amino acid sequence of FIG. 2 (SEQ ID Nos.: 2570, 2572,2574), with a proviso that the epitope is not the entire amino acidsequence of FIG. 2 (SEQ ID Nos.: 2570, 2572, 2574).
 21. The compositionof claim 20 wherein the CTL epitope comprises a polypeptide selectedfrom Tables V-XVIII, XXII, and XXIII, with a proviso that the epitope isnot the entire amino acid sequence of FIG. 2 (SEQ ID Nos.: 2570, 2572,2574).
 22. A composition of claim 1 wherein the substance comprises anantibody polypeptide epitope of the amino acid sequence of FIG. 2 (SEQID Nos.: 2570, 2572, 2574), with a proviso that the epitope is not theentire amino acid sequence of FIG. 2 (SEQ ID Nos.: 2570, 2572, 2574).23. A composition of claim 22 wherein the antibody epitope comprises apeptide region of at least 5 amino acids of FIG. 2 (SEQ ID Nos.: 2570,2572, 2574) in any whole number increment up to 875 that includes anamino acid position selected from: an amino acid position having a valuegreater than 0.5 in the Hydrophilicity profile of FIG. 5, an amino acidposition having a value less than 0.5 in the Hydropathicity profile ofFIG. 6; an amino acid position having a value greater than 0.5 in thePercent Accessible Residues profile of FIG. 7; an amino acid positionhaving a value greater than 0.5 in the Average Flexibility profile onFIG. 8; or an amino acid position having a value greater than 0.5 in theBeta-turn profile of FIG. 9, with a proviso that the epitope is not theentire amino acid sequence of FIG. 2 (SEQ ID Nos.: 2570, 2572, 2574).24. The recombinant protein of claim 23, which comprises murine antigenbinding region residues and human constant region residues.
 25. Apolynucleotide that encodes an analog peptide of claim
 19. 26. Acomposition of claim 1 wherein the substance comprises a polynucleotidethat comprises an 108P5H8-related protein coding sequence, with aproviso that the coding sequence does not encode the entire amino acidsequence of FIG. 2 (SEQ ID Nos.: 2570, 2572, 2574).
 27. The compositionof claim 26 in human unit dose form.
 28. A composition of claim 26comprising a polynucleotide from position number 44 through number 2671of FIG. 2 (SEQ ID Nos.: 2569, 2571, 2573).
 29. The composition of claim28 wherein T is substituted with U.
 30. A composition of claim 32 thatcomprises the coding sequence for the polynucleotide of FIG. 2 (SEQ IDNos.: 2569, 2571, 2573).
 31. The composition of claim 30 wherein T issubstituted with U.
 32. A composition of claim 26 comprising apolynucleotide that encodes an 108P5H8-related protein that is at least90% homologous to the entire amino acid sequence shown in FIG. 2 (SEQ IDNos.: 2570, 2572, 2574).
 33. The composition of claim 32 wherein thepolynucleotide encodes an 108P5H8-related protein that is at least 90%identical to the entire amino acid sequence shown in FIG. 2 (SEQ IDNos.: 2570, 2572, 2574).
 34. A composition of claim 26 wherein thesubstance comprises a polynucleotide that encodes at least one peptideset forth in Tables V-XVIII, XXII, and XXIII, with a proviso that theamino acid sequence of FIG. 2 (SEQ ID Nos.: 2570, 2572, 2574) is notencoded.
 35. A composition of claim 26 comprising a polynucleotide thatencodes a peptide region of at least 5 amino acids of FIG. 2 (SEQ IDNos.: 2570, 2572, 2574) that includes an amino acid position selectedfrom: an amino acid position having a value greater than 0.5 in theHydrophilicity profile of FIG. 5, an amino acid position having a valueless than 0.5 in the Hydropathicity profile of FIG. 6; an amino acidposition having a value greater than 0.5 in the Percent AccessibleResidues profile of FIG. 7; an amino acid position having a valuegreater than 0.5 in the Average Flexibility profile on FIG. 8; or anamino acid position having a value greater than 0.5 in the Beta-turnprofile of FIG. 9, with a proviso that the entire amino acid sequence ofFIG. 2 (SEQ ID Nos.: 2570, 2572, 2574) is not encoded.
 36. A compositioncomprising a polynucleotide that is fully complementary to apolynucleotide of claim
 26. 37. A composition comprising apolynucleotide that is fully complementary to a polynucleotide of claim28.
 38. A composition comprising a polynucleotide that is fullycomplementary to a polynucleotide of claim
 29. 39. A compositioncomprising a polynucleotide that is fully complementary to apolynucleotide of claim 30, in human unit dose form.
 40. A compositioncomprising a polynucleotide that is fully complementary to apolynucleotide of claim
 31. 41. A composition comprising apolynucleotide that is fully complementary to a polynucleotide of claim32.
 42. A composition comprising a polynucleotide that is fullycomplementary to a polynucleotide of claim
 33. 43. A compositioncomprising a polynucleotide that is fully complementary to apolynucleotide of claim
 34. 44. A pharmaceutical composition of claim 1wherein the substance comprises a ribozyme that cleaves a polynucleotidehaving 108P5H8 coding sequence and a physiologically acceptable carrier.45. A pharmaceutical composition of claim 1 wherein the substancecomprises a nucleic acid molecule that encodes a ribozyme that cleaves apolynucleotide having 108P5H8 coding sequence and a physiologicallyacceptable carrier.
 46. A pharmaceutical composition of claim 1 whereinthe substance comprises human T cells, wherein said T cells specificallyrecognize a 108P5H8 peptide sequence in the context of a particular HLAmolecule.
 47. A method of inhibiting growth of cancer cells thatexpresses 108P5H8, the method comprising: administering to the cells thecomposition of claim
 1. 48. A method of claim 47 of inhibiting growth ofcancer cells that express 108P5H8, the method comprising steps of:administering to said cells an antibody or fragment thereof either ofwhich specifically bind to a 108P5H8-related protein.
 49. A method ofclaim 47 of inhibiting growth of cancer cells that express 108P5H8, themethod comprising steps of: administering to said cells a vector thatencodes a single chain monoclonal antibody that immunospecifically bindsto an 108P5H8-related protein.
 50. A method of claim 47 of inhibitinggrowth of cancer cells that express 108P5H8, the method comprising stepsof: administering to said cells an 108P5H8-related protein.
 51. A methodof claim 47 of inhibiting growth of cancer cells that express 108P5H8,the method comprising steps of: administering to said cells a vectorthat comprises a polynucleotide comprising a 108P5H8-related proteincoding sequence.
 52. A method of claim 47 of inhibiting growth of cancercells that express 108P5H8, the method comprising steps of:administering to said cells an antisense polynucleotide complementary toa polynucleotide having a 108P5H8 coding sequence.
 53. A method of claim47 of inhibiting growth of cancer cells that express 108P5H8, the methodcomprising steps of: administering to said cells a ribozyme that cleavesa polynucleotide having 108P5H8 coding sequence.
 54. A method of claim47 of inhibiting growth of cancer cells that express 108P5H8 and aparticular HLA molecule, the method comprising steps of: administeringto said cells human T cells, wherein said T cells specifically recognizean 108P5H8 peptide sequence in the context of the particular HLAmolecule.
 55. A method of treating a patient who bears cancer cells thatexpress 108P5H8, the method comprising: administering to the patient thecomposition of claim
 1. 56. A method of claim 55 for treating a patientwho bears cancer cells that expresses 108P5H8, the method comprisingsteps of: administering to said patient an antibody or fragment thereofeither of which specifically binds to a 108P5H8-related protein.
 57. Amethod of claim 55 for treating a patient who bears cancer cells thatexpresses 108P5H8, the method comprising steps of: administering to saidpatient a vector that encodes an antibody or fragment thereof either ofwhich immunospecifically bind to an 108P5H8-related protein.
 58. Amethod of claim 57 for treating a patient with a cancer that expresses108P5H8, the method comprising steps of: administering to said patient avector that delivers a single chain monoclonal antibody coding sequence,whereby the encoded single chain antibody is expressed intracellularlywithin cancer cells that express 108P5H8.
 59. A method of claim 55 fortreating a patient who bears cancer cells that express 108P5H8, themethod comprising steps of: administering to said patient an108P5H8-related protein.
 60. A method of claim 55 for treating a patientwho bears cancer cells that express 108P5H8, the method comprising stepsof: administering to said patient a vector that comprises apolynucleotide comprising a 108P5H8-related protein coding sequence. 61.A method of claim 55 for treating a patient who bears cancer cells thatexpress 108P5H8, the method comprising steps of: administering to saidpatient an antisense polynucleotide complementary to a polynucleotidehaving a 108P5H8 coding sequence.
 62. A method of claim 55 for treatinga patient who bears cancer cells that express 108P5H8, the methodcomprising steps of: administering to said patient a ribozyme thatcleaves a polynucleotide having an 108P5H8 coding sequence.
 63. A methodof claim 55 for treating a patient who bears cancer cells that express108P5H8, the method comprising steps of: administering to said patient anucleic acid molecule that encodes a ribozyme that cleaves apolynucleotide having an 108P5H8 coding sequence.
 64. A method of claim55 for treating a patient who bears cancer cells that express 108P5H8and a particular HLA molecule, the method comprising steps of:administering to said patient human T cells, wherein said T cellsspecifically recognize an 108P5H8 peptide sequence in the context of theparticular HLA molecule.
 65. A method of generating a mammalian immuneresponse directed to 108P5H8, the method comprising: exposing cells ofthe mammal's immune system to an immunogenic portion of a) an108P5H8-related protein and/or b) a nucleotide sequence that encodessaid protein, whereby an immune response is generated to 108P5H8.
 66. Amethod of inducing an immune response of claim 65, said methodcomprising: providing a 108P5H8-related protein that comprises at leastone T cell or at least one B cell epitope; contacting the epitope with amammalian immune system T cell or B cell respectively, whereby the Tcell or B cell is induced.
 67. The method of claim 66 wherein the immunesystem cell is a B cell, whereby the induced B cell generates antibodiesthat specifically bind to the 108P5H8-related protein.
 68. The method ofclaim 66 wherein the immune system cell is a T cell that is a cytotoxicT cell (CTL), whereby the activated CTL kills an autologous cell thatexpresses the 108P5H8-related protein.
 69. The method of claim 66wherein the immune system cell is a T cell that is a helper T cell(HTL), whereby the activated HTL secretes cytokines that facilitate thecytotoxic activity of a cytotoxic T cell (CTL) or the antibody producingactivity of a B cell.
 70. An assay for detecting the presence of a108P5H8-related protein or polynucleotide in a biological sample from apatient who has or who is suspected of having cancer, comprising stepsof: contacting the sample with a substance of claim 1 that specificallybinds to the 108P5H8-related protein or polynucleotide, respectively;and, determining that there is a complex of the substance and108P5H8-related protein or the substance and 108P5H8-relatedpolynucleotide, respectively.
 71. An assay of claim 70 for detecting thepresence of a 108P5H8-related protein in a biological sample from apatient who has or who is suspected of having cancer, comprising stepsof: contacting the sample with an antibody or fragment thereof either ofwhich specifically bind to the 108P5H8-related protein; and, determiningthat there is a complex of the antibody or fragment thereof and108P5H8-related protein.
 72. The assay in accordance with claim 70further comprising a step of: taking a sample from a patient who has orwho is suspected of having cancer.
 73. The assay of claim 70 fordetecting the presence of an 108P5H8 polynucleotide in a biologicalsample, comprising: contacting the sample with a polynucleotide probethat specifically hybridizes to the polynucleotide of FIG. 2 (SEQ IDNos.: 2569, 2571, & 2573); and, detecting the presence of ahybridization complex formed by the hybridization of the probe with108P5H8 polynucleotide in the sample, wherein the presence of thehybridization complex indicates the presence of 108P5H8 polynucleotidewithin the sample.
 74. An assay in accordance with claim 70 fordetecting the presence of 108P5H8 mRNA in a biological sample from apatient who has or who is suspected of having cancer, said methodcomprising: producing cDNA from the sample by reverse transcriptionusing at least one primer; amplifying the cDNA so produced using 108P5H8polynucleotides as sense and antisense primers, wherein the 108P5H8polynucleotides used as the sense and antisense primers are capable ofamplifying 108P5H8 cDNA; and detecting the presence of the amplified108P5H8 cDNA.
 75. A method for monitoring 108P5H8 gene products in abiological sample from a patient who has or who is suspected of havingcancer, the method comprising: determining the status of 108P5H8 geneproducts expressed by cells in a tissue sample from an individual;comparing the status so determined to the status of 108P5H8 geneproducts in a corresponding normal sample; and, identifying the presenceof aberrant 108P5H8 gene products in the sample relative to the normalsample.
 76. A method of monitoring the presence of cancer in anindividual comprising: performing the method of claim 75 whereby thepresence of elevated gene products 108P5H8 mRNA or 108P5H8 protein inthe test sample relative to the normal tissue sample indicates thepresence or status of a cancer.
 77. The method of claim 76 wherein thecancer occurs in a tissue set forth in Table I.